Sodium butyrate ameliorates high-fat diet-induced ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Bioactive Constituents, Metabolites, and Functions

Sodium butyrate ameliorates high-fat diet-induced NAFLD through PPAR#mediated activation of # oxidation and suppression of inflammation Bo Sun, Yimin Jia, Jian Hong, Qinwei Sun, Shixing Gao, Yun Hu, Nannan Zhao, and Ruqian Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01189 • Publication Date (Web): 01 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Sodium butyrate ameliorates high-fat diet-induced NAFLD through PPARα-mediated

2

activation of β oxidation and suppression of inflammation

3

Bo Sun1,2, Yimin Jia1,2, Jian Hong2,3, Qinwei Sun1,2, Shixing Gao1,2, Yun Hu1,2, Nannan Zhao1,2,

4

Ruqian Zhao1, 2*

5

1MOE

6

Agricultural University, Nanjing 210095, P. R. China

7

2Key

8

210095, P. R. China

9

* Corresponding author: [email protected]

Joint International Research Laboratory of Animal Health & Food Safety, Nanjing

Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing

10

Tel. 00862584395047

11

Fax: 00862584398669

12

1

ACS Paragon Plus Environment


13

Abstract

14

PPARα plays a protective role against NAFLD. NaB has been shown to alleviate NAFLD, yet

15

whether and how PPARα is involved in the action of NaB remains elusive. In this study, NaB

16

administration alleviated high-fat diet-induced NAFLD in adult rats, with a decrease of hepatic

17

TG content from 108.18 ± 5.77 µg/mg to 81.34 ± 7.94 µg/mg (P < 0.05), which was associated

18

with a significant activation of PPARα. NF-κB-mediated NLRP3 signaling and pro-inflammatory

19

cytokines release were diminished by NaB treatment. NaB-induced PPARα up-regulation

20

coincided with reduced protein content of HDAC1 and promoted H3K9Ac modification on the

21

promoter of PPARα, whereas NaB-induced suppression of inflammation was linked to

22

significantly increased PPARα binding with p-p65. Conclusion: NaB acts as a histone deacetylase

23

inhibitor to up-regulate PPARα expression with enhanced H3K9Ac modification on it promoter.

24

NaB-induced PPARα activation stimulates fatty acid β oxidation and inhibits NF-κB-mediated

25

inflammation pathways via protein-protein interaction, thus contributes to amelioration of high-fat

26

diet-induced NAFLD in adult rats.

27

Key words: fatty liver, NF-κB, NLRP3, PPARα, sodium butyrate.

2


Page 2 of 34

Page 3 of 34


28

1. Introduction

29

Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in the

30

world. The main feature of NAFLD pathogenesis is the accumulation of triglyceride (TG)

31

accompanied by metabolic inflammation. It may further progress towards liver cirrhosis and

32

hepatocellular carcinoma

33

NAFLD has long been a focus of biomedical research.

34

Peroxisome proliferator-activated receptors α (PPARα) is a subtype of PPAR superfamily that is

35

highly expressed in liver. PPARα acts as a transcription factor to regulate an array of genes

36

involved in fatty acid uptake, mitochondrial fatty acid oxidation 4-5, and inflammatory response6 .

37

It has been shown that hepatocyte-specific deletion of PPARα impairs fatty acid catabolism,

38

resulting in hepatic lipid accumulation and NAFLD 7. Activation of hepatic PPARα signaling

39

significantly alleviates NAFLD by promoting mitochondria β oxidation 8. Also, PPARα is reported

40

to suppress the expression of pro-inflammatory factors by abolishing p65 binding to NF-κB

41

response element (NRE) via protein-protein interaction 6.

42

Butyrate is a short-chain fatty acid (SCFA) that naturally exists in butter and cheese 9, and can also

43

be produced endogenously by the colonic bacterial anaerobic fermentation of fiber

44

polysaccharides

45

directly acting in the intestine to inhibit fat absorption 11 or by targeting the liver to increase fatty

46

acids oxidation

47

pharmacological activities in various tissues

48

(TLR4) expression, NF-κB activation and attenuates macrophages infiltration18-19. In adipose

49

tissue, NaB alleviates obesity-induced inflammation by inhibiting NLRP3 inflammasome pathway

10.

12

1-3.

Therefore, searching for effective strategies to prevent or treat

Previous studies indicate that sodium butyrate (NaB) attenuates NAFLD by

and decrease fatty acid synthesis 13. Moreover, butyrate has anti-inflammatory 14-17.

In liver, butyrate inhibits Toll-like receptor 4

3



50

20.

51

Butyrate is reported to restore the hepatic PPARα expression suppressed by high-fat diet feeding,

52

which implicates a possible role of PPARα in mediating the NAFLD-alleviating action of butyrate

53

21.

54

a histone deacetylase inhibitor (HDACi) in the epigenetic gene regulation 22. To date, it remains

55

elusive whether PPARα is involved in the action of butyrate in alleviating fat deposition and

56

inflammation in liver and whether butyrate regulates hepatic PPARα expression through

57

modulating the status of histone acetylation on its promoter.

58

Therefore, in this study we fed rats with high-fat diet to establish NAFLD model and

59

demonstrated the efficacy of NaB in ameliorating NAFLD. Moreover, we show that

60

PPARα-mediated activation of β-oxidation and inhibition of inflammation is associated with NaB

61

action and NaB-induced transactivation of hepatic PPARα expression is related to suppressed

62

HDAC activity and hyperacetylation of PPARα promoter.

63

2. Materials and Methods

64

2.1 Chemicals

65

Chemicals, including sodium butyrate（98% purity），citric acid trisodium salt dehydrate (99.0%

66

purity), glycine, Tris-base and sodium dodecyl sulfate, were obtained from Sigma Chemical

67

Company (St Louis, MO, USA). All other reagents were commercial products of the highest

68

purity grade available.

69

2.2 Animal model and experimental protocol

70

A total of 24 male specific-pathogen-free Sprague Dawley rats (aged 6 weeks) purchased from

71

Beijing Vital River Laboratory Animal Technology Co., Ltd. were kept in the Animal Core

Butyrate exerts its biological function through different mechanisms, one of which is acting as

4


Page 4 of 34

Page 5 of 34


72

Facility of Nanjing Medical University. The animals were housed under the following typical

73

conditions: 22 ± 2°C, 50-60% humidity, 12 h light/12 h dark lighting regime. The rats were

74

allowed free access to food and water and assimilated to their environment for one week. The rats

75

were randomly divided into two groups, fed a basal diet (CON, n = 8) and a high-fat diet (HF, 45%

76

calorie from lard and sesame oil, n = 16), respectively, for 9 weeks to establish obesity and

77

NAFLD. The rats in CON group were incessantly kept on the basal diet throughout the experiment,

78

whereas the HF rats remained on the HF diet and subsequently assigned into two groups (n = 8).

79

One HF group received a vehicle for comparison with HFB, which received 300 mg/kg NaB per

80

rat via gavage every other day for 7 weeks. At the end of week 7, the rats were fasted overnight

81

and sacrificed with an anesthetic (1% barbital sodium, 500 mg/kg). Blood and liver samples were

82

harvested and stored at -80°C. The gavage dosage (300 mg/kg NaB) was determined based on a

83

previous study indicating that both 200 and 400 mg/kg could effectively alleviate insulin

84

resistance, fat accumulation, and dyslipidemia in rats with type 2 diabetes

85

procedure followed the method used in our previous study on HF-induced obese mice 24-25.

86

All procedures were approved by the Animal Ethics Committee of Nanjing Agricultural University

87

(Project No. 2012CB124703), which complied with the “Guidelines for the Ethical Treatment of

88

Experimental Animals” (2006) No. 398 provided by the Ministry of Science and Technology,

89

China.

90

2.3 Hepatic content of triglyceride and total cholesterol

91

Hepatic liver content of triglyceride (TG) and total cholesterol (Tch) was measured using

92

respective commercial assay kits (E1013 for TG and E1015 for Tch, Applygen Technologies, Inc.,

93

Beijing, China) following the manufacturer’s instruction. 5


23.

The treatment


94

2.4 Hepatic mitochondrial respiratory chain complex Ⅲ and complex V activity

95

Mitochondrial respiratory chain complex Ш and complex V was measured using respective

96

commercial assay kits (ly3 for complex Ш and ly5 for complex V; Comin Technologies, Co., Ltd.,

97

Suzhou, China) according to the manufacturer’s instruction.

98

2.5 Adenosine triphosphate (ATP) content

99

Liver ATP levels were measured using a ATP assay kit (Beyotime, China) according to the

100

manufacturer’s instructions. Luminance was measured by CLARIOstar microplate reader (BMG

101

LABTECH, Ortenberg, GER). The protein concentration was measured by BCA protein assay.

102

Data were normalized to the control group and expressed as percentage of control levels.

103

2.6 Determination of cytokines by ELISA

104

The contents of cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor

105

necrosis factor α (TNFα) in liver were determined using the ELISA kits (ANG-E11546R for IL-1β,

106

ANG-E11755R for IL-6 and ANG-E11546R for TNFα, Angle Gene Bioengineering Co., Ltd.,

107

Nanjing, China) according to the manufacturer’s protocol. Detection range: IL-1β from 1.6 ng/L to

108

80 ng/L, IL-6 from 8 ng/L to 150 ng/L, TNFα from 15 ng/L to 300 ng/L. The intra- and inter-assay

109

coefficients of variation were less than 9% and 15% respectively.

110

2.7 Histopathology and immunofluorescence

111

The liver tissues were cut and fixed with a 4% paraformaldehyde, and were embedded in paraffin.

112

Sections (4 μm) were stained with hematoxylin-eosin (HE) or Oil Red O (ORO) to investigate

113

architecture of the liver and hepatic lipid droplets. Stained slides were scanned with the

114

Pannoramic SCAN II and images were captured with 3DHISTECH software (3DHISTECH Ltd.

115

Budapest, Hungary). Immunofluorescence was used for checking the expression of CD68 (M1 6


Page 6 of 34

Page 7 of 34


116

macrophage marker) and CD206 (M2 macrophage marker). Briefly, liver sections (4 μm) were

117

deparaffinized with xylene, followed by antigen retrieval by heating in citrate buffer (10 mmol/L)

118

at 95°C for 15 min. The following primary antibodies were used: anti-CD68 (ab955, Abcam, USA)

119

and CD206 (ab64693, Abcam, USA). The Alexa Fluor® 488 conjugated goat anti-rat IgG for

120

CD68 (ab150157, Abcam, USA) and Alexa Fluor® 488 conjugated donkey anti-rabbit IgG for

121

CD206 (ab150073, Abcam, USA) were used respectively as the second antibody. DAPI was used

122

as a marker for cell nuclei.

123

2.8 RNA isolation and quantitative real-time PCR

124

Total RNA was isolated from 30 mg liver samples by using 1 mL of TRIzol reagent (Invitrogen,

125

USA). Up to 1 µg of the RNA samples was reverse-transcribed following the protocol provided by

126

the manufacturer (Vazyme Biotech, Nanjing, China). The cDNA was diluted at 1:25 of which 2 μL

127

was used as template in PCR reactions on a real-time PCR system (Mx3000P, Stratagene, USA).

128

Moreover, 18S was chosen as a reference gene to normalize the mRNA abundance of target genes.

129

All primers were made by Generay Biotech and listed in Table 2. The 2-ΔΔCt method was used to

130

analyze real-time PCR data.

131

2.9 Protein extraction and Western blot analysis

132

Total protein was extracted from 50 mg frozen liver samples as previously described26. Protein

133

concentrations were measured using the BCA Protein Assay Kit (No. 23225, Thermo Scientific,

134

USA) according to the instructions provided by the manufacturer. Protein (40 µg/lane) was used

135

for electrophoresis on a 7.5% or 10% sodium dodecyl sulfate (SDS) polyacrylamide gel

136

electrophoresis and then transferred onto a polyvinylidene fluoride membrane. Membranes were

137

blocked in 5% milk and then incubated in primary and secondary antibodies. Western blot analysis 7



138

for PPARα (BS1689, Bioworld, USA, diluted 1:500) and carnitine palmitoyltransferase 1 (BS7744,

139

Bioworld, USA, diluted 1:1000) was performed, using tubulin (BS1699, Bioworld, USA, diluted

140

1:5000) as a reference protein for normalization of loading and transfer efficiency. Western blot

141

analysis was conducted for cytochrome c oxidase subunit 1 (COX1) (BS70809, Bioworld, USA,

142

diluted 1:500), cytochrome c oxidase subunit 4 (COX4) (BS2186, Bioworld, USA, diluted 1:500),

143

F4/80 (70076s, Cell Signaling Technology, USA, diluted 1:1000), and CD206 (ab64693, Abcam,

144

USA, diluted 1:1000) by using GAPDH (AP0063, Bioworld, USA, dilute 1:5000) as a reference

145

protein. The same GAPDH image was used for the normalizations of six target proteins, including

146

the phospho–inhibitor of nuclear factor kappa-B kinase alpha/beta (p-IKKα/β) (2697s, Cell

147

Signaling Technology, USA, diluted 1:1000), inhibitor of nuclear factor kappa-B kinase γ (IKKγ)

148

(sc-166700, Santa Cruz, USA, diluted 1:200), p-p65 (ab6503, Abcam, USA, diluted 1:1000),

149

NLRP3 (ab214185, Abcam, USA, diluted 1:1000), cleaved caspase-1 (ab179515, Abcam, USA,

150

diluted 1:1000), and histone deacetylases 1 (HDAC1) (BS6485, Bioworld, USA, diluted 1:500) as

151

they were detected in the same blot. Proteins of distinct sizes, such as cleaved caspase-1 (20 kDa),

152

NLRP3 (110 kDa), and p-IKKα/β (85 kDa), were considerably well separated and could be

153

detected using their respective membrane slices probed with their respective antibodies. Stripping

154

and re-probing were applied for proteins of the similar size. Images were captured using the

155

VersaDoc 4000MP system (Bio-Rad, USA), and band density was analyzed using the software

156

Quantity One (Bio-Rad, USA).

157

2.10 Chromatin immunoprecipitation assay

158

Chromatin immunoprecipitation (ChIP) was analyzed as described in a previous study, with

159

several modifications27. Frozen liver samples (300 mg) in liquid nitrogen were homogenized and 8


Page 8 of 34

Page 9 of 34


160

then resuspended in PBS containing Protease Inhibitor Cocktail (No. 11697498001; Roche).

161

Protein was cross-linked to DNA by adding formaldehyde to a final concentration of 1%. The

162

reaction was stopped subsequently with 2.5 mol/L glycine at room temperature. The mixture

163

resulting from the aforementioned procedure was centrifuged (5000 rpm for 5 min at 4°C). Pellets

164

were rinsed with PBS and homogenized in an SDS lysis buffer containing protease inhibitors.

165

Crude chromatin preparations were sonicated on ice to yield DNA fragments of 150-500 bp in

166

length and pre-cleared with salmon sperm DNA-treated protein G agarose beads (40 μL, 50%

167

slurry, sc-2003, Santa Cruz, USA). The mixture of pre-cleared chromatin preparations and 2 μg of

168

primary antibody (H3 lysine 9 acetylation, ab4441, Abcam, USA) were incubated overnight at

169

4°C. A negative control was included with normal IgG. Protein G agarose beads (40 μL, 50%

170

slurry, sc-2003; Santa Cruz) were added to capture the immunoprecipitated chromatin complexes.

171

Finally, DNA fragments were released from the immunoprecipitated complexes by reverse

172

cross-linking at 65°C for 1 h, and quantitative real-time PCR was used to quantify the fragments

173

of target gene promoters with specific primers (Table 2) using purified immunoprecipitated DNA

174

as the template.

175

2.11 Co-immunoprecipitation

176

Co-immunoprecipitation was conducted as previously described, with slight modifications

177

Total protein amounting to 500 µg was incubated with 20 μL of 50% protein A/G plus beads for 2

178

h at 4°C and then centrifuged at 7,500 g for 1 min. The supernatants were incubated with 3 µg

179

p-p65 antibodies and rotated overnight at 4°C. Subsequently, 20 μL of agarose beads was

180

incubated with protein-antibody complexes for 10 h at 4°C. The agarose beads were washed with

181

cold PBS after centrifugation, and the immunoprecipitated proteins were run on 10% 9


28.


182

SDS-polyacrylamide gel for Western blot analysis.

183

2.12 Statistical analysis

184

All data are presented as mean ± SEM. One-way ANOVA with a Bonferroni post hoc test was

185

applied to estimate the differences among the three groups. P < 0.05 was considered statistically

186

significant.

187

3. Results

188

3.1 NaB reduces high-fat diet-induced body weight gain and hepatic lipid deposition

189

Compared to the rats fed control diet, the rats fed high-fat diet for 9 weeks had significantly (P

Sodium butyrate ameliorates high-fat diet-induced ... - ACS Publications

Recommend Documents