Effects of Dietary Protein from Different Sources on Biotransformation

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Food Safety and Toxicology

Effects of dietary protein from different sources on biotransformation, antioxidation and inflammation in rat liver Xuebin Shi, Xisha Lin, Yingying Zhu, Yafang Ma, Yingqiu Li, Xing-lian Xu, Guanghong Zhou, and Chunbao Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01717 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Journal of Agricultural and Food Chemistry

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Effects of dietary protein from different sources on biotransformation, antioxidation

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and inflammation in rat liver

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Xuebin Shi, Xisha Lin, Yingying Zhu, Yafang Ma, Yingqiu Li, Xinglian Xu,

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Guanghong Zhou*, Chunbao Li*.

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Key Laboratory of Meat Processing and Quality Control, MOE; Key Laboratory of

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Meat Processing, MOA; Jiangsu Synergetic Innovative Center of Meat Processing

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and Quality Control; Nanjing Agricultural University; Nanjing, 210095, P.R. China

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*Corresponding author: Dr Guanghong Zhou, [email protected]

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Dr Chunbao Li, [email protected]

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ACS Paragon Plus Environment


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Abstract: In this work, the effects of different sources of meat protein on liver

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metabolic enzymes were investigated. Rats were fed for 90 days semisynthetic diets in

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which casein was fully replaced by isolated soybean, fish, chicken, pork or beef

13

proteins. Then liver proteomics was performed using iTRAQ and LC−ESI−MS/MS.

14

The results indicated that intake of meat protein diets significantly reduced the protein

15

levels of CYP450s, GSTs, UGTs, and SULTs compared to the casein and soybean

16

protein diet groups. The total antioxidant capacity and lipid peroxidation values did

17

not differ between four meat protein diet groups and the casein diet group. However,

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GSH activity in the fish, chicken, and beef protein groups were significantly higher

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than those of the casein and soybean protein groups. Beef protein diet significantly

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upregulated the expression of immune-related proteins. The Keap1-Nrf2-ARE

21

signaling pathway was suggested to involve the diet-mediated regulation of

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biotransformation, inflammation and redox status.

23 24

Keywords: dietary

protein,

rat liver,

biotransformation, inflammation,

antioxidant

25

2


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Introduction

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Meat is a good source of essential amino acids, fatty acids and micronutrients.

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Evaluating the associations between meat intake and human health has becoming a

29

hot topic in recent years. 1 However, excessive intake of meat, the same as other foods,

30

will increase the risk to metabolic disorders.2 Although some safety evaluation studies

31

have shown that overdoses of heme iron, benzopyrene, and heterocyclic amines in

32

meat may be harmful to the subjects,

33

endogenous components of meat, e.g., protein, on human health.

34

3-5

few studies are available on the effects of

In a short term feeding study, Song et al.

6

found that meat proteins increased

35

the gene expression of redox and immune response in rat liver. In another study, Zhu

36

et al.7 found that intake of meat proteins downregulated the expression of

37

lipopolysaccharidebinding protein (LBP) in rat serum than those fed with soybean

38

protein. These studies indicated beneficial functions of meat proteins. However, its

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underlying mechanism is unclear.

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The liver is the central metabolic organ for the detoxification of many

41

xenobiotics. Biotransformation reactions and play a critical role in xenobiotics

42

metabolism in the liver. CYP450s, GSTs, UGTs, and SULTs are involved in such a

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process. Hepatoprotective effects of natural compounds have been frequently

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attributed to their antioxidant properties and the ability to mobilize endogenous

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antioxidant defense system.8 Some studies showed that hepatoprotective effects of

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natural compounds9 and drug10 such as antioxidants, were related to biotransformation

47

capacity. Therefore, we hypothesize that the dietary proteins may alter metabolic

48

enzymes and metabolites in liver, then adjust the liver biotransformation capacity, and

49

further mediate the antioxidant and anti-inflammatory responses. In this study, the rat

50

liver proteomics was studies through the method of iTRAQ labeling and 3



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LC−ESI−MS/MS to explore the possible molecular mechanisms resulted by the

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different protein sources.

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Material and methods

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Diets and Animals. Diets were prepared as described by Zhu et al.7 Briefly, the

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animal meat( pork longissimus dorsi muscle, beef longissimus dorsi muscle, chicken

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pectoralis major muscle and fish muscle) were chopped and cooked in a 72 °C water

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bath for 0.5 h. The cooked meat was chilled, freeze-dried and pulverized, and then

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immersed in organic solvent to remove the intramuscular fat. Casein and soybean

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proteins were purchased, respectively. Soybean protein isoflavones was removed by

60

alcohol extraction. In accordance with recommendation of the American Institute of

61

Nutrition (AIN-93),

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(Nantong, China). Except owing different protein resources, all diets were balanced

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for total energy content (4056.0 kcal/kg) and macronutrient content (protein 177g/kg,

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fat 70 g/kg, and carbohydrate 679.5 g/kg).

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All

11

animals

various groups of rat diet were made by Jiangsu Teluofei, Inc.

were

treated

under

the

guidelines

of

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Experimental Animal Ethical Committee of Nanjing Agricultural University. A total of

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66 male SD rats (117 g ± 10 g) from Zhejiang Experimental Animal Center were fed

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in a specific pathogen-free environment. After 1-week adaption period, the rats were

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arranged randomly to six formulated diets with six proteins for 90 days. The rats fed

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with ad libitum and adequate water in the individual cages and stay in a stable

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environment with temperature (20.0±0.5°C), humidity (60±10%) and a 12 h light-dark

72

cycle. After 90 days of feeding, the rats were sacrificed after 4 h fasting. Liver

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samples were obtained, and stored at -80 °C until analysis. Each protein diet group

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has 11 biological samples (n=11).

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Protein extraction. Liver proteins were extracted according to the procedures of 4


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Song et al with minor modified.12 Briefly, 0.1g liver samples were homogenized for

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60 s in 1 mL iced protein lysis buffer (Beyotime P0013K) containing 1% protease

78

inhibitors (SIGMA P8340) and 1% protease inhibitors (SIGMA P0044). The

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homogenates were centrifuged (4˚C, 16000g) for 1 h and the pellets were discarded.

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The supernatant and chilled acetones with 10% (v/v) TCA (1:5) were mixed and

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stored in -20 °C for 4 h. The samples were centrifuged (4˚C, 16000g) for 15 min and

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the pellets were collected. After washing with chilled acetone, the pellets dissolved in

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UA buffer(8M urea+0.1M/HCl pH8.5), sonicated and centrifuged (4˚C, 16000g) for

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15 min and the supernatant used for the quantification of protein concentration.

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A certain volume of protein samples (150 µg protein) were mixed with 400 µL

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UA buffer in the ultra-filtration tube and centrifuged (4˚C, 14000g) for 15 min. The

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samples were mixed with 200 µL UA buffer and 50 mM DTT, and then incubated at

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60 °C. Subsequently, 50 mM iodoacetamide (IAM) was added and incubated for 45

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min in dark at room temperature. The supernatant was mixed with 100 µL dissolution

90

buffer (from iTRAQ kit). Then the mixture was centrifuged (4, 30000g) for 15 min

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and collected the pellets. This step was repeated twice to remove iodoacetamide. The

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pellets were performed with trypsin digested (protein: trypsin was 30:1) at 37 ˚C for

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16 h, then centrifuged and quantified by NanoDrop spectrophotometer.

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ITRAQ labeling and high pH reverse phase fractionation. Peptides were

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labeled by 8-plex iTRAQ reagent kit (Applied Biosystems) based on the

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manufacture’s protocol. The peptides samples mixed with iTRAQ reagent and

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incubation in 20˚C for 2 h. Then all the labeled samples were pooled and

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vacuum-dried. A total of eleven peptide mixtures were prepared.

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The labeled peptides fractionation was performed by the high pH reverse phase

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fractionation chromatography. Firstly, the labeled peptides were reconstituted in 5



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100µL of buffer A (98% acetonitrile, 2% H2O), and loaded onto the column. The

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peptides were eluted at a flow rate of 0.2 mL/min using a gradient of 97% to 3%

103

buffer A, and 3% to 97% buffer B (2% acetonitrile, 98% H2O) for 60 min. After that,

104

elution was measured the absorbance at 214 nm. The eluted peptides were collected

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every 1 min and separated into 60 fractions. According to the differences in the

106

fraction polarity (fraction collection time), these fractions were mixed into 8 samples

107

and vacuum-dried.

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Nano LC-MS/MS conditions. The identification of samples was performed as

109

described previously13. After dissolving in 0.1% formate, the peptides (1.5g) was

110

loaded onto an column (Acclaim PepMap100 C18, 100 µm×2 cm, 5 µm, 100 Å,

111

Thermo Scientific). Subsequently, the peptides eluted on an analytical column

112

(Acclaim PepMap® RSLC, C18,75 µm×10 cm, 3µm, 100 Å, Thermo Scientific) by a

113

gradient of 97% to 3% buffer A (0.1% formate), and 3% to 97% buffer B (80%

114

acetonitrile, 0.1% formate) at 300 nL/min flow rate over 160 min. The MS/MS

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conditions were in accordance with previous study. 13

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Data analysis. Data analysis was handled with Proteome Discoverer Software

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(Thermo Fisher Scientific, Waltham, MA, USA). Protein was identified by Sequest

118

HT engine against the UniprotKB Rattus Norvegicus database (update to January,

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2016). Searching parameters were in accordance with previous study.13 The selection

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of differential protein based on the average of |Fold Change (FC)| ≥2.0 in meat protein

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diet groups (fish: F; chicken: C; pork: P; beef: B) compared to the other two group

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(soybean: S; casein: L).

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Hepatic oxidative stress analysis. Liver samples (0.2 g) were added into 1.8 mL

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physiological saline and homogenized for 60 s. The homogenates were centrifuged

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(4˚C, 3500g) for 15 min. The supernatants were collected and protein concentration 6


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was measured. The enzymatic activities of T-AOC, SOD and GSH-PX; the

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abundances of GSH and MDA were analyzed with commercial kits (Nanjing

128

Jiancheng Bioengineering Institute, Nanjing, China).

129

RT-PCR. Total RNA was extracted from liver samples using a commercial RNA

130

extraction kit (Takara, No.9796). The Nanodrop 2000 spectrophotometer was used to

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measure the purity and quantity of total RNA at 260 and 280 nm. cDNA was reverse

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transcribed using Prime Script RT Master Mix Kit (Takara, No.RR036A). The

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RT-PCR reactions were run using SYBR Premix Ex Taq Kit (TaKaRa, No.RR420A).

134

Primers were presented in Supplemental Table 1. Relative mRNA levels were

135

calculated using the 2−∆∆Ct method. GAPDH was applied as reference gene to

136

determine the levels of Nrf2, Keap1, TNFα, NFκB, IL1β, Sult1a1, Sult1b1, Ugt1a1

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and Gstµ2.

138

Bioinformatics and statistical analysis. The protein expression matrix was

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generated by DataMerge2 and normalized, and abundance-differential proteins were

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evaluated by the t test. OmicsBean (http://www.omicsbean.cn) was used for

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bioinformatics analysis of differential proteins. The MeV program was used to make

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heat maps of differential proteins. The data was analyzed by Duncan’s multiple range

143

test with a significance level set at P < 0.05.

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Results

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The general difference in liver metabolism in rats varied with meat protein

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diets. Comparative proteomics indicated that liver protein abundance was changed by

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intake of meat proteins by 3% to 10%, and the increasing order was fish, chicken,

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beef and pork protein diet groups. In general, the proteomics data revealed that

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various metabolic processes and liver biotransformation were regulated. However, the 7



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differential proteins involving biotransformation were the same between four meat

151

protein diet groups and control groups (Supplemental table 2). Of these differential

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proteins, Ugt2b, Gstα1, Ste belong to phase II enzymes. Annexin VI (Anxa6) is

153

implicated in membrane-related events along exocytotic and endocytotic pathways.

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Carboxylesterase 1D（Ces1d）involves the metabolism of xenobiotics and of natural

155

substrates. Carbonic anhydrase 3（Ca3） is a major participant in the liver response to

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oxidative stress. paraoxonase arylesterase 1 (Pon1) is capable of hydrolyzing a broad

157

spectrum of organophosphate substrates and lactones. Compared to the casein group,

158

the four meat protein diet groups had higher abundance of T-kininogen 1 (Map1) that

159

is a liver-specific acute phase glycoprotein and plays an important role in coagulation

160

cascade. Protein-protein interaction (PPI) indicated that Ugt2b37 (RGD:3937) was the

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core node for several biological pathways, including the shared cytochrome p450

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pathway with Gstα1 (Fig.1), which mediated exogenous substance, drug metabolism

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and chemical carcinogenic metabolism. Ugt2b37 and estrogen sulfotransferase 3 (Ste)

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also involved the steroid hormone biosynthesis pathway. At the same time,

165

glutathione and ascorbate metabolism were regulated by Ugt2b and Gstα1, which are

166

also an important part of the body's antioxidant activity. Therefore, the present study

167

focused on the effects of different meat protein diets on liver biotransformation,

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inflammation and antioxidant in rats.

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Dietary proteins regulated biotransformation enzymes in rat liver. Meat

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protein diets significantly altered the enzymes of liver biotransformation in rats. In

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particular, several enzymes involved liver oxidation and binding reactions (Fig. 2).

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ALDH is a kind of multifunctional protease that is dependent on NAD(p)+, which is

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generally expressed in the mitochondria and other organelles in liver tissues.

174

Compared with the casein group, the abundances of Aldh2, Aldh3a2, Aldh6a1, 8


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Aldh7a1, Aldh8a1were lower in meat protein groups. Similarly, compared with

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soybean protein group, Aldh1b1, Aldh1l1, Aldh2, Aldh4a1, Aldh6a1, Aldh8a1 had

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lower abundance in meat protein groups (P < 0.05, Fig.2).

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In beef and chicken protein groups, Aldh1b1 and Aldh1l1 were downregulated as

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compared to the soybean protein group. Aldh1b1 has catalytic activity on the short or

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medium chain aliphatic aldehyde, aromatic aldehyde and lipid peroxidation products,

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including 4-hydroxylaldehyde and malondialdehyde. Aldh1l1, i.e., 10-formacyl

182

tetrahydrofolate dehydrogenase, participates in the biological synthesis of purines and

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may affect the DNA replication and repair. Aldh2, a critical enzyme involving

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acetaldehyde oxidation and lipid peroxidation, exhibited a lower abundance in meat

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protein diet groups than that in the casein diet group. Aldh3a2, i.e., fatty aldehyde

186

dehydrogenase was downregulated in pork and chicken protein diet groups. This

187

enzyme is composed of fatty alcohol: NAD redox enzyme complex and fatty alcohol

188

dehydrogenase and is involved in oxidation of fatty alcohols into fatty acids. Aldh4a1,

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i.e., pyrroline-5-carboxylate (P5C), was downregulated in beef, chicken and fish

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protein diet groups. The enzyme involves proline degradation by catalyzing P5C into

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neurotransmitter glutamate to prevent its accumulation. Aldh4a1 may play a role in

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DNA repair

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dehydrogenase that is associated with valine and pyrimidine catabolism, was

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downregulated in the pork protein diet group. Aldh7a1 was downregulated in beef,

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chicken and pork protein diet groups. This enzyme plays a major role in lysine

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catabolism. Aldh8a1 has metabolic activity in several important aldehyde, such as

197

succinate half aldehyde and glutaraldehyde, exhibited a lower abundance in meat

198

protein diet groups than that in the soybean protein diet group.

199

and

cell

survival.14

Aldh6a1,

methylmalonate-

semialdehyde

Binding reaction is an important form of liver biotransformation, including 9



200

several CYP450s, UGTs, GSTs and SULTs. In the present study, the pork and beef

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protein diet groups had significantly lower abundances of CYP450s proteins,

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including Cyp2c11, Cyp2d1 and Cyp2d26. Cyp2c subtribes account for 20% of the

203

total liver CYP450s. Of them, rat Cyp2c11 is homologous with human Cyp2c9, and

204

responsible for the metabolism of most of the nervous system drugs. 15-16 Rat Cyp2d1

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is homologous with human Cyp2d6, and may be involved in dietary nutrient and drug

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metabolism, but less involved in the activation of carcinogenic substances and

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teratogenic substances.17-18

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UGTs play a major role in the conjugation and subsequent elimination of

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potentially toxic xenobiotics and endogenous compounds. Hydroxyl and carboxyl

210

compounds can be conjugated with glucuronic acid and be inactivated. In the present

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study, Ugt1a and Ugt2b subtypes were downregulated in meat protein diet groups

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compared to the casein and soybean protein groups.

213

GSTs catalyze the conjugation of reduced glutathione to exogenous or

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endogenous hydrophobic electrophiles, and the products will be combined with bile

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acids and excreted with feces.19 In the present study, GSTs, in particular to Gstµ2 and

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Gst1a, were downregulated in the meat protein diet groups, compared to the casein

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and soybean protein groups.

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SULTs utilize 3'-phospho-5'-adenylyl sulfate as sulfonate donor to catalyze the

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sulfate conjugation of catecholamines, phenolic drugs and neurotransmitters, and they

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also have estrogen sulfotransferase activity. In this study, the abundances of Sult1a1,

221

Sult1b1 and Sult1c3 were significantly lower in meat protein diet groups than the

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casein and soybean protein diet groups.

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RT-PCR was applied to verify diet-induced changes of gene expression of the

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above enzymes (Fig.3). The mRNA levels of Sult1a1, Sult1b1, Gstµ2 and Ugt1a1 10


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were lower in meat protein diet groups than the casein or soybean protein diet groups

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(P < 0.05).

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Protein diets affected rat liver inflammation and other related proteins.

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Inflammation is a part of the complex biological responses to harmful stimuli, such

229

as pathogens,

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involving immune cells, blood vessels, and molecular mediators. During early

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inflammation, histamines, peptides and proteases are involved in the regulation of

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vascular permeability. In the present study, several proteins related to inflammation,

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immunity, and antioxidant responses were regulated by protein diets (P < 0.05, Fig.4).

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Map1 was upregulated by the four meat protein diets. Map1 can be used as a sensitive,

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rather than specific inflammatory biomarker.20-21 T-kininogen 2 (P08932) was

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upregulated in the beef protein group compared to the soybean protein diet group.

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Galectins have a special affinity for β-galactoside and play a role in cell adhesion and

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inflammation.22 In the present study, the abundances of galectin 1 (Lgals1) and

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galectin 3 (Lgals3) were much higher in the four meat protein diet groups, in

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particular for the pork and beef protein groups than those in the casein group. Lgals3

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can mediate the activation of T cells and promote the function of antigen

242

presentation.23

damaged

cells,

or

irritants,

and

is

a

protective

response

243

On the other hand, the meat protein diet groups showed lower abundances of

244

Icam1, Crp, Pgrmc1 and Psme1 than the soybean protein diet group, and lower

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abundances of Pgrmc1, Pgrmc2 and Crp than the casein diet group (P < 0.05). Icam1,

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intercellular adhesion molecule 1, participates in the acute inflammatory response of

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antigen stimulation. Membrane-associated progesterone receptor components 1 and 2

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(Pgrmc1, Pgrmc2) can be combined with steroid hormones and has many reported

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cellular functions (interaction with CYP450s). Crp, C-reactive protein, displays 11



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several functions associated with host defense, and it promotes agglutination,

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bacterial capsular swelling, and phagocytosis. Crp can also interact with DNA and

252

histones and scavenge nuclear material released from damaged circulating cells.

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Psme1(Proteasome activator subunit 1) involves activating NFκB. Pycard, S100A9

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and Mtdh were much lower in the pork and chicken protein diet groups than those in

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the soybean protein or casein groups. Pycard has cysteine-type endopeptidase

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activity involved in apoptotic process. S100A9 is a calcium- and zinc-binding

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protein which plays a prominent role in the regulation of inflammatory processes and

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immune response. Mtdh may activate NFκB transcription factor.

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Several immunity related proteins, e.g., P20761 M0RAV0 D3ZPL2 and

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A0A0G2JW41, were found to highly abundant in beef protein diet group compared to

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the casein diet group (P < 0.05). In addition, meat protein diet groups also showed

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higher abundance of Ig gamma-2B chain C region that participates in the classical

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antibody-mediated complement activation.

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In terms of antioxidants, pork and beef protein diets induced higher levels of

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peroxidase 5 (Prdx5) compared to the casein diet group (P < 0.05). Pork protein diet

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also induces higher level of SOD1 compared to the soybean protein diet group (P
0.05),

272

but were significantly lower than that of the soybean protein diet group (P < 0.05). In

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addition, the MDA level was not significantly different (P > 0.05) between the four

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meat protein diet groups and the casein diet group, but the values were much lower 12


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for the soybean protein diet group (P < 0.05). Of the four meat protein diet groups, the

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pork protein diet group had the lowest MDA level (P < 0.05).

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GSH-PX and SOD play a critical role in protecting against oxidative damage by

278

reducing lipid hydroperoxide to their corresponding alcohols and free hydrogen

279

peroxide to water. The fish and chicken protein diet groups had higher GSH-PX

280

activities than the soybean, beef and pork protein diet groups (P < 0.05). Similarly, the

281

fish and chicken protein diet groups had highest values of SOD activity and the pork

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and beef diet groups had the lowest (P < 0.05). The fish, chicken and beef protein diet

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groups had higher values of GSH activity than the other three groups (P < 0.05).

284

Protein diets altered inflammation index in rat liver. To evaluate the

285

inflammation and immunity status, the mRNA levels of several inflammatory factors,

286

i.e., NFκB, TNFα, IL1β, Nrf2 and Keap1 were measured. There was no significant

287

difference in NFκB mRNA level between four meat protein groups and casein or

288

soybean protein diet groups (P > 0.05, Fig. 6), however, the values were higher for the

289

fish and beef protein diet groups than that of the pork protein diet group (P < 0.05).

290

The average values of TNFα mRNA level were numerically higher for the four meat

291

protein diet groups than the casein diet group, but the differences were not statistically

292

significant (P > 0.05). The chicken and pork protein diet groups had lower values of

293

IL1β mRNA level than the casein and soybean protein diet groups (P < 0.05). The

294

average value of Nrf2 mRNA level was the highest for the fish protein diet group and

295

the lowest for the pork protein diet group. Keap1 was highly expressed in the fish

296

protein diet group (P < 0.05), but there was no significant difference among the other

297

diet groups (P > 0.05).

298

Discussion

299

Protein diets and liver biotransformation. Dietary protein metabolism is firstly 13



300

catalyzed by CYP450s by adding one or more hydroxyl groups to a non-polar

301

substance that is converted into an electrophilic or polar-activated exogenous

302

substance. Then the polar groups can be connected by GSTs, UGTs, and SULTs with

303

activated exogenous substances and their water solubility will increase. Dietary

304

protein, fibers, phytochemicals and specially processed cereals have been reported to

305

increase the activities of CYP450s, GSTs, UGTs, and SULTs.24-27 In the present study,

306

intake of meat proteins downregulated CYP450s, GSTs, UGTs, and SULTs in rat liver

307

at the mRNA and protein levels. The differences in nutrient metabolism between

308

human and rat livers could be related to the food source. The heterologous substances

309

in plant protein diets stimulate the high expression of phase I and II enzymes. The

310

animal protein diet induced relatively low levels of these enzymes, mainly due to the

311

fact that animal muscle protein (main dietary protein) is consistent with the body

312

composition and nutrient requirements of humans and experimental animals.

313

The chaperonin Keap1 has functions to regulate the uncoupling of Nrf2 in the

314

cytoplasm and the binding of Nrf2 to ARE in the nucleus, which involves the

315

activation of GSTs.28 In the present study, Nrf2 and Keap1 were differentially

316

regulated in fish and pork protein diet groups, indicating that Keap1-Nrf2-ARE

317

pathway could play a critical role in the diet-induced regulation of CYP450s, GSTs,

318

UGTs, and SULTs.

319

The response of CYP450s and GSTs to the metabolism of xenobiotics from diets

320

and drugs is related to the induction or prevention of diseases.29-30 For example,

321

estrogen can be hydroxylated by Cyp1a1 to form 4-OH-E, 2-OH-E and 16-OH-E.

322

These metabolites are then converted into semi-quinones and quinones, which can be

323

covalently combined with purines to form 4-OH-E1-N3A and 4-OH-E1-N7G. This

324

will produce iso-bases on the DNA.31 The point mutations in the iso-base sites have 14


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become potential malignant transformations.32 The quinones can be inactivated by

326

GSTs or reduced to hydroxylated estrogens by reductases. Hydroxylated estrogens can

327

be conjugated with sulfate by sulfotransferase (such as Ste) and the conjugated

328

products can be excreted. In the present study, the meat protein diet groups had low

329

levels of CYP450s and sulfotransferase, indicating that intake of meat protein diets

330

reduced the estrogen and exogenous substances binding reaction as compared to the

331

casein diet. Correspondingly, the meat protein diet groups had relatively low

332

abundance of estrogen membrane receptor protein (Pgrmc1 and Pgrmc2).

333

Protein diets and liver antioxidants. The living organisms have enzymatic and

334

non-enzymatic antioxidant systems. In the present study, the chicken and fish protein

335

diet groups had higher levels of enzymatic antioxidants than the soybean protein diet

336

group, while the pork and beef protein diet groups had lower activities than the casein

337

group. However, the SOD and Prdx5 protein levels were higher in the pork protein

338

diet group. The inconsistency between the enzymatic activity and protein levels could

339

be attributed to the different levels of cofactors (e.g. metal ions). GSH plays an

340

important role in the non-enzymatic antioxidant system, while other studies showed

341

that rice protein can decrease oxidative stress in rat by regulating GSH

342

metabolism.33-34 Similarly, meat proteins significantly increased GSH levels in this

343

study.

344

Diets alter endogenous antioxidant capacity by adjusting the ROS scavenging

345

ability.

346

greatly different amino acid composition from casein and soybean proteins, which

347

may affect gene expression of metabolic enzymes in rat liver. For example, amino

348

acid-derived (Aldh4a1, Aldh6a1 and Aldh7a1), lipid-derived (Aldh1b1, Aldh2,

349

Aldh3a2 and Aldh8a1), and nucleic acid-derived aldehyde metabolic enzymes

35

Protein source is the only variable in the present study. Meat proteins have

15



350

(Aldh1l1, Aldh4a1 and Aldh6a1) were downregulated by meat protein diets. ALDHs

351

catalyze the irreversible oxidation of endogenous aldehydes to produce the organic

352

acids and protect the body from the toxicity of aldehydes.36 And thus, the abundances

353

of ALDHs may reflect the aldehyde level and redox state in the body. Simultaneously,

354

Ca3, as the liver response to oxidative stress and Pon1, an enzymatic protection of

355

low density lipoproteins against oxidative modification were significantly lower in

356

meat protein diet groups and implied a low oxidation state.

357

It is well known that Nrf2-Keap1-ARE signaling pathway regulates cell

358

protection and antioxidant stress. Surya et al. observed that 4-hydroxy-2-nonenal in

359

the colon could activate Nrf2-dependent antioxidation, and regulate the resistance of

360

preneoplastic cells upon exposure to fecal water of hemoglobin- and beef-fed

361

rats.37 Song et al. indicated that dietary proteins from different sources regulated

362

antioxidant processes through the nuclear factor Nfe2l2 (known as Nrf2).38 Li et al.

363

showed that rice protein upregulated endogenous antioxidant (GSTs) and

364

detoxification function by activating the Nrf2 pathway.39 In the present study, the Nrf2

365

mRNA level was upregulated in the fish protein diet group, which was consistent with

366

the high level of antioxidant activities. The lower level of Nrf2 mRNA in the pork

367

protein diet group was in accordance with lower antioxidant enzyme activities and

368

lower abundance of GSTs, UGTs, and SULTs. When estradiol is bound to estrogen

369

receptor (ER), the activated ER might recruit corepressors to Nrf2, which would

370

reduce Nrf2-ARE-regulated transcription of detoxifying enzymes.40 Therefore, the

371

difference in estrogen metabolism between casein and meat protein diet groups

372

reflects, to a certain context, the difference in detoxification enzyme activities.

373

Protein diet and liver inflammation. Inflammation is a defensive response of a

374

living tissue to an injury factor. Inflammation may change vascular permeability and 16


Page 16 of 34

Page 17 of 34


375

immune response. In the present study, Map1, which involves the regulation of

376

vascular permeability, was upregulated in meat protein diet groups. This indicates that

377

diet-induced changes of vascular permeability could occur in the pro-inflammatory

378

phase. C-reactive protein (Crp) is an acute-phase protein, whose levels were reduced

379

in the meat protein diet group. The acute inflammatory response was not increased by

380

meat protein diet.

381

NFκB and TNFα play a role in immune response and inflammation.41-42 In the

382

present study, no significant difference was observed in NFκB and TNFα mRNA

383

levels between the meat protein diet groups and the control groups. However, the

384

meat protein diet groups had lower abundances of acute inflammatory response

385

(Icam1), and the regulatory proteins of NFκB including Mtdh, Psme1 and Pycard.

386

This indicates that intake of meat proteins does not increase the burden of

387

inflammation. Moreover, Zhu et al. (2015) found that intake of meat proteins

388

decreased the levels of serum lipopolysaccharide binding protein (LBP) compared to

389

the soybean protein group. Lipopolysaccharides are gut-derived endotoxins and can

390

activate NFκB-regulated inflammatory signaling pathways.7 Donkey milk was

391

observed to reduce proinflammatory signs (TNFα and LPS), probably by Nrf2

392

pathway.43 Coincidentally, in the present study, the mRNA expression of Nrf2 and

393

NFκB in each group were consistent and were relatively higher in the casein group

394

than the meat protein groups. The inflammatory level of meat protein was

395

comparable or lower in casein diet.

396

Dietary proteins play a critical role in the development of the immune system,

397

which act as antigenic stimulators of serum immunoglobulins.44 In ruminants,

398

soybean and fish proteins were shown to have a significant effect on the abundance of

399

serum immunoglobulin.45 Song et al. found that fish proteins diets reduced immune 17



400

responses, and chicken and beef altered complement/coagulation-pathways.6,12

401

Therefore, we suggest that plant protein diet regulated antigen presenting via

402

xenobiotic metabolism (Anxa6, Ces1d). Casein diet regulated antigen presenting via

403

estrogen (Pgrmc1 and Pgrmc2). Meat protein diet regulated antigen presenting via

404

heme homeostasis (Lgals1, Lgals3). Beef protein diet has a higher level of heme

405

homeostasis, so immunoglobulin was upregulated. Therefore, dietary proteins alter

406

the immune response from antigen presenting to immunoglobulin expression.

407

Abbreviation used

408

ALDH, aldehyde dehydrogenase; ARE, antioxidant response element; CYP450s,

409

cytochrome P450 enzymes; GSH, reduced glutathione; GSH-PX, glutathione

410

peroxidase; GST, glutathione S-transferases; Nrf2, nuclear factor-erythroid 2-related

411

factor 2; Keap1, Kelch-like ECH-associated protein 1; MDA, malonaldehyde; NFκB,

412

nuclear factor kappa-light-chain-enhancer of activated B cells; T-AOC, total

413

antioxidant capacity; TNFα, tumor necrosis factor α; SOD, superoxide dismutase;

414

SULT, sulfotransferase; UGT, UDP-glucuronosyltransferase.

415

Acknowledgments

416

We would like to thank Chen Dai, Yun Hu, He Li, Li Li, Chong Wang from

417

Nanjing Agricultural University for their help during animal feeding and sampling.

418

This work was financially supported by the National Natural Science Foundation

419

(34171600, 31530054) and PAPD.

420

Supporting Information

421 422

The Supporting Information is available free of charge on the ACS Publications website at DOI:

423

Supplemental Table 1 Primer sequences of target and reference genes for

424

real-time PCR. Supplemental Table 2 Differentially expressed proteins in the rat liver 18


Page 18 of 34

Page 19 of 34


425

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426

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reduces

energy

efficiency

and

improves

24


antioxidant

and

Page 25 of 34


563

FIGURE CAPTIONS

564

Fig.1 Protein-protein interaction analysis of similar difference of expressed protein

565

between each meat proteins and casein in the rat liver. Note: The node represents a

566

protein in PPI analysis of liver proteome. Each quarter represents a corresponding

567

comparison, P: L, pork protein versus casein; C: L, chicken protein versus casein; B:

568

L, beef protein versus casein; F: L, fish protein versus casein. Green node color

569

represents lower abundance in meat protein diet groups when compared to casein

570

diet. The node size is proportional to the connection of each signaling pathway. The

571

squares represent signaling pathways (KEGG Term). The colors of squares indicate

572

the significance (log p-value) of changes of signaling pathway with blue for high and

573

yellow for low.

574 575

Fig.2 Effect of dietary meat proteins on expression of rat liver biotransformation

576

related protein. NOTE: The significant difference in protein expression was

577

consistent with the green concentration in the figure. F: S, fish protein versus

578

soybean protein; C: S, chicken protein versus soybean protein; P: S, pork protein

579

versus soybean protein; B: S, beef protein versus soybean protein; F: L, fish protein

580

versus casein; C: L, chicken protein versus casein; P: L, pork protein versus casein;

581

B: L, beef protein versus casein.

582 583

Fig.3 Effect of dietary casein, soybean and meat proteins on rat liver phase II

584

enzymes related genes expression. Values are represented as means ± SEM from 25



585

nine experiments (n=9). Groups were compared by one-way ANOVA followed by

586

Duncan’s multiple range tests. Letters a−c represent significant differences between

587

diet groups (p < 0.05). L, casein; S, soybean protein; F, fish protein; C: chicken

588

protein; P, pork protein; B, beef protein.

589 590

Fig.4 Effects of dietary casein, soybean and meat proteins on rat liver immunity,

591

inflammation and antioxidation related protein. The colors of suares indicate the

592

directions of changes of proteins with red for up-regulation and blue for

593

down-regulation. F: S, fish protein versus soybean protein; C: S, chicken protein

594

versus soybean protein; P: S, pork protein versus soybean protein; B: S, beef protein

595

versus soybean protein; F: L, fish protein versus casein; C: L, chicken protein versus

596

casein; P: L, pork protein versus casein; B: L, beef protein versus casein.

597 598

Fig.5 Effect of dietary casein, soybean and meat proteins on rat liver antioxidant

599

indicators. Values are represented as means ± SEM from nine experiments (n=9).

600

Groups were compared by one-way ANOVA followed by Duncan’s multiple range

601

tests. Letters a−c represent significant differences between diet groups (p < 0.05). L,

602

casein; S, soybean protein; F, fish protein; C: chicken protein; P, pork protein; B,

603

beef protein.

604 605

Fig.6 Effect of protein diets on rat liver inflammation and antioxidation related genes

606

expression. Values are represented as means ± SEM from three experiments (n=9). 26


Page 26 of 34

Page 27 of 34


607

Groups were compared by one-way ANOVA followed by Duncan’s multiple range

608

tests. Letters a−c represent significant differences between diet groups (p < 0.05). L,

609

casein; S, soybean protein; F, fish protein; C: chicken protein P, pork protein; B, beef

610

protein

27



Fig.1

28


Page 28 of 34

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Fig.2

29



Fig.3

30


Page 30 of 34

Page 31 of 34


Fig.4

31



Fig.5

32


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Fig.6

33



TOC

34


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Effects of Dietary Protein from Different Sources on Biotransformation

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