Abstract

Simple Summary

   Taurine has a wide range of biological functions in vertebrates but
   does not participate in protein synthesis. Recent studies showed
   taurine exhibits an important role in the regulation of fat metabolism
   in fish and has the effect of reducing liver fat accumulation in
   high-fed fish. However, it is still unclear how taurine exerts the
   effect and the underlying metabolic mechanism of taurine intervention.
   In this study, we conducted an experiment to investigate the
   fat-lowering effect of taurine on orange-spotted groupers at the
   proteomic level. Furthermore, we performed an integrated analysis of
   transcriptomics and proteomics and excavated the key genes and key
   proteins involved in the regulation of liver fat metabolism, in an
   attempt to better understand the intrinsic connection between the
   transcriptional and translational levels and interpret the trajectories
   in terms of the biological phenomena in a more comprehensive way after
   taurine intervention on high-fat fed orange-spotted groupers.

Abstract

   In order to understand the intervention effect of taurine on liver fat
   deposition induced by high fat intake in the orange-spotted grouper
   (Epinephelus coioides), we performed proteomic analysis and association
   analysis with previously obtained transcriptomic data. Three isoproteic
   (47% crude protein) diets were designed to contain two levels of fat
   and were named as the 10% fat diet (10F), 15% fat diet (15F), and 15%
   fat with 1% taurine (15FT). The 10F diet was used as the control diet.
   After 8 weeks of feeding, the 15F diet exhibited comparable weight
   gain, feed conversion ratio, and hepatosomatic index as the 10F diet,
   but the former increased liver fat content vs. the latter. Feeding with
   the 15FT diet resulted in an improvement in weight gain and a reduction
   in feed conversion ratio, hepatosomatic index, and liver fat content
   compared with feeding the 15F diet. When comparing liver proteomic data
   between the 15F and 15FT groups, a total of 133 differentially
   expressed proteins (DEPs) were identified, of which 51 were upregulated
   DEPs and 82 were downregulated DEPs. Among these DEPs, cholesterol
   27-hydroxylase, phosphatidate phosphatase LPIN, phosphatidylinositol
   phospholipase C, and 6-phosphofructo-2-kinase were further screened out
   and were involved in primary bile acid biosynthesis, glycerolipid
   metabolism, the phosphatidylinositol signaling system, and the AMPK
   signaling pathway as key DEPs in terms of alleviating liver fat
   deposition of taurine in high-fat fed fish. With the association
   analysis of transcriptomic and proteomic data through KEGG, three
   differentially expressed genes (atp1a, arf1_2, and plcd) and four DEPs
   (CYP27α1, LPIN, PLCD, and PTK2B) were co-enriched into five pathways
   related to fat metabolism including primary bile acid synthesis, bile
   secretion, glycerolipid metabolism, phospholipid D signaling, or/and
   phosphatidylinositol signaling. The results showed that dietary taurine
   intervention could trigger activation of bile acid biosynthesis and
   inhibition of triglyceride biosynthesis, thereby mediating the liver
   fat-lowering effects in high-fat fed orange-spotted grouper. The
   present study contributes some novel insight into the liver
   fat-lowering effects of dietary taurine in high-fat fed groupers.

   Keywords: taurine, liver fat metabolism, proteomics, omics association
   analysis, Epinephelus coioides

1. Introduction

   As is well known, fat is an essential nutrient for normal growth and
   development of fish due to its cell structural framework and the fact
   that it provides fuel for cellular metabolism [[34]1]. Moreover, fat is
   highly used by fish [[35]2], and the energy utilization efficiency of
   the digested fat is approximately 80% (64% for digested protein and 58%
   for digested carbohydrates, respectively) [[36]3]. Fat also has a
   protein-sparing effect in fish [[37]4,[38]5,[39]6]. As a result, this
   drives fish farmers to pursue excessive use of fat as an effective
   energy source in aquafeeds to reduce the consumption of high-quality
   proteins for fuel, promoting feed utilization [[40]7]. However, the
   feeding practice of fish has shown that prolonged intake of high-fat
   feed could cause visceral fat accumulation and fatty liver of fish
   [[41]8] and, in severe cases, metabolic disorders including fatty liver
   syndrome [[42]9,[43]10,[44]11]. Therefore, high-fat feed-induced fatty
   liver, the chronic hepatic disease relevant to nutritional metabolic
   syndrome, has become one of the important issues in current intensive
   aquaculture. In order to find an effective prevention and control
   method to address this threat, there is a need to have a deep
   understanding of the high-fat feed induced fatty liver in fish and its
   mechanism.

   Taurine, a conditionally indispensable amino acid of farmed fish,
   displays a valuable potential application in solving this problem
   [[45]12,[46]13]. It is now clear that taurine has the potential to
   control fatty liver by relieving fat metabolism disorders of fish with
   dietary taurine intervention [[47]14,[48]15]. The fat-lowering effect
   of taurine has been confirmed with effective taurine intervention on
   fish fed high-fat diets in recent years
   [[49]12,[50]13,[51]16,[52]17,[53]18,[54]19]. However, it is still
   unclear how taurine regulates the fat-lowering effect and what role it
   plays in the fat metabolism of fish. On this issue, our research team
   conducted a series of studies to investigate the intervention effects
   of taurine in vitro and in vivo and its regulatory mechanism in
   high-fat fed orange-spotted grouper (Epinephelus coioides) via
   transcriptomics, lipidomics, and metabolomics in recent years
   [[55]20,[56]21,[57]22,[58]23]. Data from our recent lipidomic analysis
   show that the reduction in liver fat accumulation via dietary taurine
   addition may be realized through decreasing the contents of TGs
   containing 18:2n-6 at the sn-2 and sn-3 positions and through promoting
   the anti-inflammatory capacity of groupers [[59]20]. In another study,
   the taurine-conjugated BAs have a higher ability to accelerate fat
   emulsification and absorption than glycine-conjugated and other BAs in
   the fish species [[60]23]. Moreover, our recent studies in vivo and in
   vitro using transcriptomics showed that the reduction in liver fat
   accumulation was attributed to the fact that dietary taurine addition
   enhanced the synthesis of endogenous taurine in the liver, accelerated
   BA transport and insulin secretion, thus promoting fatty acid
   β−oxidation efficiency [[61]21,[62]22].

   In the present study, we conducted an experiment regarding taurine
   intervention in high-fat fed orange-spotted grouper. With these
   results, we further investigated the fat-lowering effect of taurine on
   the fish species and elucidated its regulatory mechanism in fat
   metabolism at the proteomic level. In addition, we performed an
   integrated analysis of transcriptomics and proteomics based upon our
   existing transcriptomic and proteomic data and excavated the key genes
   and key proteins involved in the regulation of liver fat metabolism, in
   an attempt to better understand the intrinsic connection between the
   transcriptional and translational levels and interpret the trajectories
   in terms of the biological phenomena in a more comprehensive way after
   taurine intervention on high-fat fed orange-spotted grouper
   [[63]24,[64]25]. This study provides some new insights into the
   prevention and treatment of nutritional metabolic diseases (fatty liver
   syndrome) in fish.

2. Materials and Methods

2.1. Experimental Diets

   The optimal levels of taurine and fat in feed were 1.0% and 10%,
   respectively, for the normal growth of orange-spotted grouper in our
   previous research and those of others [[65]26,[66]27]. Thus, in this
   experiment, three isoproteic (47% crude protein) semi-purified diets
   were prepared using casein and gelatin as the main protein ingredients
   and fish and soy oils and soy lecithin as the major fat ingredients, as
   previously described in our recent research [[67]22], that is, control
   diet (10% fat diet), high-fat diet (15% fat diet), and high-fat diet
   with 1% taurine (15% fat diet with 1% taurine), designated 10F, 15F,
   and 15FT, respectively ([68]Table 1). The mixed powder feeds were made
   into sinking pellets, dried, and then stored at −20 °C until use,
   according to our previous practice [[69]12].

Table 1.

   Ingredients and proximate composition of experimental diets (on an
   as−fed basis, %).
   Ingredients Diets (Fat Level/Taurine Level)
   10F (10/0) 15F (15/0) 15FT (15/1)
   Casein:gelatin = 4:1 50 50 50
   Shrimp meal 4 4 4
   Corn starch 25 25 25
   Oil blend (fish oil:soy oil = 1:1) 6 10 10
   Soy lecithin 4 4 4
   Premix 0.8 0.8 0.8
   Ca(H[2]PO[4])[2] 2 2 2
   Microcrystalline cellulose 7.2 3.2 2.2
   Sodium alginate 1 1 1
   Taurine 0 0 1
   Nutrient level (analyzed values)
   Dry matter 91.18 90.24 90.38
   Crude protein 46.55 46.87 46.56
   Crude lipid 10.44 14.79 14.89
   Taurine 0.04 0.04 0.98
   [70]Open in a new tab

2.2. Growth Trial

   The grouper juveniles were obtained from a local hatchery (Zhangpu
   county, Fujian, China). Prior to the start of the trial, the fish were
   maintained with a commercial diet (46.1% crude protein and 9.6% crude
   fat) in a three-week acclimation. A total of 270 groupers with an
   initial wet weight of about 10.5 g were randomly distributed into nine
   tanks within a recirculating aquaculture system at a water flow rate of
   8 L/min per tank. The groupers of nine tanks were arranged into three
   treatments, each with triplicate 300 L tanks at a stock density of 30
   fish per tank. The groups of triplicate tanks were hand-fed twice daily
   (8:30 and 18:30) across a feeding period of 8 weeks. Excess feed was
   collected 30 min after each meal to determine feed intake. During the
   feeding period, the daily rearing management followed our previous
   practice [[71]11].

2.3. Sample Collection

   At the end of the 8-week feeding trial, five fish were randomly sampled
   from each tank and sacrificed with an overdose of MS-222 solution
   (tricaine methanesulfonate, Sigma-Aldrich Shanghai Trading Co., Ltd.,
   Shanghai, China), followed by fish count and batch weighing, and were
   recorded on a wet weight basis to determine percent weight gain and
   feed conversion ratio. After completing the weighing, liver was removed
   to calculate the hepatosomatic index (HSI) and pooled by tank in a tube
   for determining liver fat content. The same batch of five fish per tank
   were randomly caught and dissected to aseptically remove livers and
   were pooled into one tube by tank and then stored at −80 °C for the
   subsequent extraction of protein.

2.4. Protein Digestion and Peptide TMT Labeling

   The volume ratio of liver tissue to lysis buffer SDT in the homogenate
   was one to four. SDT (4% (w/v) SDS, 100 mM Tris/HCl, pH = 7.6, 0.1M
   DTT) buffer was used for liver sample lysis and protein extraction. The
   supernatant (protein solution) was collected. Briefly, 200 μg of
   protein solution per sample was added to a dithiothreitol solution
   (DTT, Sigma-Aldrich Shanghai Trading Co., Ltd., Shanghai, China) to
   make a mixed solution with a final concentration of 5 mmol/L, and the
   reaction was maintained at 56 °C for 30 min, followed by the addition
   of iodoacetamide (IAA, Sigma-Aldrich Shanghai Trading Co., Ltd.,
   Shanghai, China), to make a mixed solution with a final concentration
   of 11 mmol/L, and was then kept at room temperature for 30 min in the
   dark. The reaction was terminated with triethylamine-carbonate buffer
   solution (TECS). The amount of protein in the liver sample was
   quantified with the BCA Protein Assay Kit (Bio-Rad., New York, NY,
   USA). Protein digestion by trypsin was performed according to the
   filter-aided sample preparation (FASP) procedure described by Matthias
   Mann [[72]28]. The digest peptides of each liver sample were desalted
   on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed
   ID7 mm, volume 3 mL, Sigma-Aldrich Shanghai Trading Co., Ltd.,
   Shanghai, China) and then concentrated via vacuum centrifugation and
   reconstituted in 40 µL of 0.1% (v/v) formic acid [[73]29]. The
   resulting peptide mixture of each liver sample (100 μg) was labeled
   using TMT (tandem mass tag) reagent according to the manufacturer’s
   instructions (Thermo Scientific, Xiamen, China).

2.5. High-PH Reversed-Phase Peptide Fractionation

   Peptides that have been labeled with the TMT labeling kit, and then
   mixed in equal parts. The gradient elution separation of aliquots of
   labeled peptides for each liver sample was performed using the high-pH
   reversed-phase peptide fractionation kit (Thermo Scientific). The
   specific procedures refer to Zhang’s description [[74]30]. Firstly,
   column equilibration was achieved using acetonitrile and 0.1%
   trifluoroacetic acid. Then, the labeled peptide mixture was added with
   pure water, followed by centrifugation at low speed for desalination.
   Finally, step-gradient elution of column-bound peptides was performed
   with increasing concentrations of high-pH acetonitrile solution. Each
   eluted peptide sample was then vacuum dried. The lyophilized peptide
   sample was resolved in 12 μL of 0.1% TFA, and the peptide concentration
   was determined at OD280.

2.6. LC-MS/MS Analysis

   LC-MS/MS data collection refers to the method in [[75]30]. Briefly, a
   HPLC liquid-phase system (ASY-nLCTM 1200 nm version) was used for the
   separation of each sample. Buffers A and B were 0.1% formic acid and
   0.1% formic acid acetonitrile (84% acetonitrile), respectively. After
   the column equilibrium was achieved with 95% buffer solution A, the
   sample was injected via the autosampler to the loading column (Thermo
   Scientific Acclaim PepMap100, 100 µm × 2 cm, nanoViper C18) and
   separated through the analytical column (Thermo Scientific EASY column,
   10 cm, ID 75 µm, 3 µm, C18-A2) with a flow rate of 300 nL/min.

   After completion of chromatographic separation, the peptides were
   analyzed using a Q-Exactive mass spectrometer (Thermo Scientific). The
   detection conditions were set as follows: detection mode: positive ion;
   parent ion scan range: 300–1800 m/z; first order mass spectrometry
   resolution: 70,000 at 200 m/z; automatic gain control target setting:
   100,000; maximum injection time: 50 ms; and dynamic exclusion time: 60
   s.

   Mass charge ratios of polypeptides and polypeptide fragments were
   collected according to the following methods: 20 fragment maps were
   collected for each full scan. The specific parameter settings were as
   follows: MS2 Activation Type: HCD (higher-energy collision-induced
   dissociation); isolation window: 2 m/z; resolution of the secondary
   mass spectrometry: 17,500 at m/z 200; normalized collision energy: 30
   eV; and lower fill ratio: 0.1%.

2.7. Database Search and Data Analysis

   After completion of the LC-MS/MS analysis, the data in raw files were
   checked in databases through the software Mascot2.2 (Matrix Science,
   London, UK) and the software Proteome Discoverer1.4 (Thermo Fisher
   Scientific, San Jose, CA, USA), and the TMT labeling quantitative
   analysis was performed. The database search method described by Lin was
   performed [[76]31]. Briefly, the MS raw data for each sample were
   searched using the Mascot engine (Matrix Science, London, UK; version
   2.2) embedded into Proteome Discoverer 1.4 software (Thermo Electron,
   San Jose, CA, USA) for identification and quantitation analysis. The
   Mascot search parameters and instructions are as follows: Max missed
   cleavages: 2; fixed modifications: Carbamidomethyl (C), iTRAQ 4/8 plex
   (N-term), iTRAQ 4/8 plex (K), TMT 6/10/16 plex (N-term), TMT 6/10/16
   plex (K); variable modifications: oxidation (M), Ox iTRAQ 4/8 plex (Y),
   TMT 6/10/16 plex (Y); peptide mass tolerance: ±20 ppm; fragment mass
   tolerance: 0.1 Da; database pattern: decoy; and peptide FDR ≤ 0.01.

   The differentially expressed proteins (DEPs) were filtered under the
   criteria of p-value < 0.05 and fold change (FC) > 1.2-fold for
   upregulation or less than 0.83 for downregulation [[77]32]. The number
   of up- and downregulated DEPs of liver in the comparison group was
   finally obtained.

2.8. Bioinformatics Analysis

   Blast2GO ([78]https://www.blast2go.com/) was used to annotate functions
   of DEPs by GO, which was categorized into three main groups: biological
   process, molecular function, and cellular component [[79]33]. The DEPs
   were numbered after GO function annotation of all DEPs [[80]34]. The
   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was
   performed on DEPs through the database for the KEGG pathway by using
   the KAAS (KEGG Automatic Annotation Server) online service tool to
   associate the KEGG ORTHOLOG (KO) numbers of the DEPs to the KEGG
   pathway [[81]35].

2.9. Parallel Reaction Monitoring (PRM) Analysis

   To further verify the reliability of DEPs with TMT analysis, eight
   randomly selected DEPs were subjected to PRM validation ([82]Table 2).
   Peptide information was imported into the software Xcalibur4.3 (Thermo
   Fisher Scientific, San Jose, CA, USA) for PRM method setup.
   Approximately 1 μg of peptide taken from each sample and 20 fmol of
   labeled peptide (PRTC: GISNEGQNASIK) were mixed. According to the
   method in [[83]36], gradient separation of peptides was performed using
   a high-performance liquid chromatography system with buffer solution A:
   0.1% formic acid aqueous solution and buffer solution B: 0.1% formic
   acid acetonitrile aqueous solution (84% acetonitrile). After separation
   of peptides, the target peptides were analyzed via PRM mass
   spectrometry using a Q-Exactive HF mass spectrometer (Thermo
   Scientific). The specific parameters were as follows: primary MS scan
   range: 300–1800 m/z; MS resolution: 60,000 (m/z 200); automatic gain
   control target setting: 3,000,000; maximum injection time: 200 ms;
   analysis time: 60 min; detection mode: positive ions; 20 MS2 scans at
   each primary MS scan; PRM scans (MS2 scans), isolation window: 1.6 Th;
   MS resolution: 30,000 (m/z 200); MS2 activation type: HCD; and
   normalized collision energy: 27 ev. A total of 9 samples from three
   groups were subjected to PRM detection, and the PRM raw files were
   analyzed on the software Skyline 3.5.0 (MacCoss Lab, University of
   Washington, Seattle, WA, USA).

Table 2.

   Protein expression profile in the TMT detection mode.
   Protein ID Protein Abbreviation Protein
   Description FC TMT Pattern PRM
   Pattern
   TR5402_c1_g1_ORF CYP27α1 Cholesterol 27-hydroxylase 1.564 up up
   TR511_c4_g1_ORF PFKFB1 6-phosphofructo-2-kinase 1.269 up up
   TR314_c8_g1_ORF PLCD Phosphatidylinositol phospholipase C 0.792 down
   down
   TR944_c0_g1_ORF_1 ABAT 4-aminobutyrate aminotransferase 1.393 up up
   TR1786_c0_g1_ORF GNAL Guanine nucleotide-binding protein G subunit
   alpha 0.803 down down
   TR1499_c4_g2_ORF GANC Neutral alpha-glucosidase C 0.804 down nd
   TR2721_c0_g1_ORF ENPP1_3 Ectonucleotide pyrophosphatase 0.785 down down
   TR244239_c0_g1_ORF PCBD 4a-hydroxytetrahydrobiopterin dehydratase 1.319
   up up
   [84]Open in a new tab

2.10. Association Analysis of Transcriptomic and Proteomic Data

   Based on the transcriptomic data previously obtained in our laboratory
   and the proteomic data currently obtained, special attention needs to
   be paid to the DEGs and DEPs enriched through the KEGG database, as
   well as their related pathways. The association analysis of DEGs and
   DEPs was performed to identify any common pathways between DEGs and
   DEPs, or to find pathways that are correlated upstream and downstream,
   or the pathways that have common metabolites. To this end, a network
   diagram was constructed to demonstrate the joint participation of DEPs
   and DEGs in liver fat metabolism after taurine intervention on a
   high-fat fed grouper.

2.11. Statistical Analysis

   Data are presented as means ± SD with n = 3 for growth weight, feed
   conversion ratio, and liver fat content, while hepatosomatic index data
   are presented as means ± SD with n = 15. Significant differences among
   dietary treatments were analyzed with one-way ANOVA and
   Student–Neuman–Keuls multiple comparison test after data were tested
   for normality and homogeneity of variance with the Kolmogorov–Smirnov
   test and Levene’s test in SPSS Statistics 22.0 (SPSS, Michigan Avenue,
   Chicago, IL, USA). Student’s t-test was applied for comparison between
   the 15F diet and 15FT diet. The processing of histograms was performed
   using GraphPad prism 9.0 (San Diego, CA, USA) software. A p-value <
   0.05 was considered statistically significant.

3. Results

3.1. Growth Performance and Liver Fat Contents

   The growth performance of groupers is shown in [85]Figure 1. There was
   no difference in weight gain, feed conversion ratio, and hepatosomatic
   index between the 10F and 15F diets (p > 0.05). However, the 15FT diet
   resulted in improved weight gain and reduced feed conversion ratio and
   hepatosomatic index compared with the 15F diet (p < 0.05).

Figure 1.

   [86]Figure 1
   [87]Open in a new tab

   Effects of experimental diets on growth performance and liver fat
   contents of groupers. Groups: 10F, 10% fat diet; 15F, 15% fat diet;
   15FT, 15% fat diet with 1% taurine. Weight gain (%) = 100 × (final body
   weight − initial body weight)/initial body weight; feed conversion
   ratio = feed intake/wet weight gain; hepatosomatic index (%) = 100 ×
   (liver weight/wet body weight); values for WG, FCR, and liver fat
   content are presented as the means ± SD (n = 3 tanks); values of
   hepatosomatic index are presented as the means ± SD (n = 15 fish);
   values on the bar with different lowercase letter superscripts indicate
   significant differences (p < 0.05); statistical analysis was performed
   using one-way ANOVA, followed by Student−Neuman−Keuls multiple
   comparison test.

   The liver fat content in the 15F diet was significantly higher (p <
   0.05) than that in the 10F diet. Fish fed the 15FT diet had lower (p <
   0.05) liver fat content compared with those fed the 15F diet, and
   exhibited comparable liver fat content to those of fish fed the 10F
   diet (p > 0.05).

3.2. Proteome Profiling

   Compared with the 10F diet, a significantly higher liver content was
   observed in the 15F diet fed fish, and the distinct intervention effect
   of taurine on the 15FT diet fed fish was also observed. Therefore, we
   chose 15F and 15FT as the comparison group to analyze the subsequent
   fat metabolic response after taurine intervention at the proteomic
   level.

   A total of 133 DEPs were filtered by comparing the 15FT and 15F groups,
   of which 51 proteins were significantly upregulated and 82 proteins
   were significantly downregulated in the taurine group compared with the
   non-taurine group ([88]Figure 2).

Figure 2.

   [89]Figure 2
   [90]Open in a new tab

   Volcano plot for identified and quantified proteins in the present
   study. Red and blue dots indicate proteins that were differentially
   expressed, up− or downregulation, respectively, in the 15% fat diet
   with 1% taurine group versus the 15% fat diet group.

3.3. Bioinformatics Analysis

   GO analysis included biological process, molecular function, and
   cellular component. As shown in [91]Figure 3, the dominant
   subcategories were cellular process and metabolic process in the
   category of biological processes, the binding activity and catalytic
   activity appeared dominant in the category of molecular function, and
   the cell and cell part showed the highest percentage in cellular
   component.

Figure 3.

   [92]Figure 3
   [93]Open in a new tab

   GO annotation statistics of differentially expressed proteins in 15%
   fat diet with 1% taurine group versus 15% fat diet group.

   The DEPs were subjected to a KEGG pathway enrichment analysis. Results
   are shown in [94]Figure 4. In the present study, we mainly focused on
   metabolic pathways directly related to fat metabolism, such as primary
   bile acid synthesis and glycerophospholipid metabolism, although many
   DEPs were enriched in metabolic pathways such as calcium signaling and
   purine metabolism.

Figure 4.

   [95]Figure 4
   [96]Open in a new tab

   Bubble plot of KEGG pathway enrichment in 15% fat diet with 1% taurine
   group versus 15% fat diet group.

3.4. Protein Pathways Related to Fat Metabolism after Taurine Intervention

   After KEGG pathway enrichment analysis of DEPs, the major protein
   pathways that were involved in fat metabolism included the following:
   glycerophospholipid metabolism, primary bile acid biosynthesis,
   phospholipase D signaling pathway, AMPK signaling pathway, etc. The
   results are shown in [97]Table 3. Thus, the liver protein network
   related to fat metabolism was constructed by comparing the 15FT diet
   versus the 15F diet ([98]Figure 5).

Table 3.

   Identification result of differentially expressed proteins related to
   fat metabolism in 15% fat diet with 1% taurine group versus 15% fat
   diet group.
   Protein ID Map Name Protein Description FC p-Value Expression Pattern
   TR5402_c1_g1_ORF Primary bile acid biosynthesis Cholesterol
   27-hydroxylase 1.564 0.0181 Up
   TR854_c2_g1_ORF_1 Glycerolipid metabolism/Glycerophospholipid
   metabolism/mTOR signaling pathway Phosphatidate phosphatase LPIN 0.812
   0.0114 Down
   TR147_c2_g1_ORF Glycerophospholipid metabolism/MAPK signaling
   pathway-yeast Glycerol-3-phosphate dehydrogenase 0.726 0.0307 Down
   TR2191_c0_g1_ORF Glycerophospholipid metabolism Phosphatidylserine sn-1
   acylhydrolase 0.816 0.0393 Down
   TR969_c0_g1_ORF_1 Sphingolipid metabolism Ceramide synthetase 0.81
   0.0217 Down
   TR3417_c0_g1_ORF Sphingolipid signaling pathway transcription factor
   0.822 0.00281 Down
   TR1786_c0_g1_ORF Calcium signaling pathway Guanine nucleotide-binding
   protein G(olf) subunit alpha 0.803 0.0297 Down
   TR1499_c4_g2_ORF Galactose metabolism Neutral alpha-glucosidase C 0.804
   0.0303 Down
   TR61997_c0_g2_ORF Sphingolipid signaling pathway/FoxO signaling pathway
   Mitogen-activated protein kinase 0.796 0.0119 Down
   TR43919_c0_g1_ORF Phospholipase D signaling pathway/Calcium signaling
   pathway Focal adhesion kinase 2 0.797 0.0053 Down
   TR52940_c0_g1_ORF mTOR signaling pathway Calcium binding protein 0.767
   0.0443 Down
   TR511_c4_g1_ORF AMPK signaling pathway 6-phosphofructo-2-kinase 1.269
   0.0153 Up
   TR2721_c0_g1_ORF Starch and sucrose metabolism Ectonucleotide
   pyrophosphatase 0.785 0.0359 Down
   TR244239_c0_g1_ORF Folate biosynthesis 4a-hydroxytetrahydrobiopterin
   dehydratase 1.319 0.0351 Up
   TR944_c0_g1_ORF_1 beta-Alanine metabolism 4-aminobutyrate
   aminotransferase 1.393 0.0101 Up
   TR76930_c0_g1_ORF Citrate cycle Malate dehydrogenase 0.801 0.0486 Down
   TR65955_c0_g1_ORF Endocytosis Arf-GAP with SH3 domain 0.825 0.0437 Down
   TR314_c8_g1_ORF Phosphatidylinositol signaling system/Calcium signaling
   pathway Phosphatidylinositol phospholipase C 0.792 0.0403 Down
   [99]Open in a new tab

Figure 5.

   [100]Figure 5
   [101]Open in a new tab

   Network diagram of proteins related to fat metabolism in the liver of
   groupers after taurine intervention. CYP27A1, cholesterol
   27-hydroxylase; PFKFB1, 6-phosphofructo-2-kinase; ABAT, 4-aminobutyrate
   aminotransferase; PCBD, 4a-hydroxytetrahydrobiopterin dehydratase;
   PLCD, phosphatidylinositol phospholipase C; LPIN, phosphatidate
   phosphatase LPIN; SREBP1, sterol regulatory element-binding protein 1;
   FAS, fatty acid synthase; ACC1, acyl-CoA carboxylase1; mTOR, mammalian
   target of rapamycin; PLD1_2, phospholipase D1_2; FBP,
   fructose-1,6-bisphosphatase I; AMPK, AMP-activated protein kinase;
   RAPTOR, regulatory-associated protein of mTOR; RHOA, Ras homolog gene
   family, member A; PTK2B, Protein Tyrosine Kinase 2 Beta; HSD3B7,
   hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid
   delta-isomerase 7; AKR1D1, aldo-keto reductase family 1, member D1;
   CDCA, chenodeoxycholic acid; CA, cholic acid; TCDCA,
   taurochenodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; TCA,
   taurocholic acid; GCA, glycocholic acid.

3.5. Validation of TMT Results with PRM

   Eight DEPs were subjected to PRM analysis to validate the reliability
   of TMT results. As is shown in [102]Figure 6, dietary taurine addition
   upregulated the liver expression levels of cholesterol 27-hydroxylase
   (CYP27A1), 6-phosphofructo-2-kinase (PFKFB1), 4-aminobutyrate
   aminotransferase (ABAT), and 4a-hydroxytetrahydrobiopterin dehydratase
   (PCBD) but downregulated the liver expression levels of
   phosphatidylinositol phospholipase C (PLCD), neutral alpha-glucosidase
   C (GANC), and ectonucleotide pyrophosphatase (ENPP1_3); however,
   guanine nucleotide-binding protein G subunit alpha (GNAL) was not
   affected by dietary taurine addition. There were the same trends of
   liver expression changes for the above seven proteins subjected to PRM
   analysis, except GNAL, as those subjected to TMT analysis after
   high-fat fed fish were intervened with taurine. These results indicated
   that the high-throughput data through TMT analysis was of high quality
   and the screening results were reliable in this study.

Figure 6.

   [103]Figure 6
   [104]Open in a new tab

   PRM verification of the expression levels of target proteins in the
   liver. Values are expressed as the means ± SD (n = 3). Asterisks (*, **
   and ***) represent significant differences with p < 0.05, p < 0.01, and
   p < 0.001, respectively. CYP27A1, cholesterol 27-hydroxylase; PFKFB1,
   6-phosphofructo-2-kinase; PLCD, Phosphatidylinositol phospholipase C;
   GNAL, guanine nucleotide-binding protein G subunit alpha; GANC, neutral
   alpha-glucosidase C; ABAT, 4-aminobutyrate aminotransferase; ENPP1,
   ectonucleotide pyrophosphatase; PCBD, 4a-hydroxytetrahydrobiopterin
   dehydratase.

3.6. Association Analysis of DEGs and DEPs

   There were three DEGs (atp1a, plcd, and arf1_2) and four DEPs (CYP27A1,
   LPIN, PLCD, and PTK2B) that were associated with five KEGG pathways
   such as primary BA synthesis, bile secretion, glycerolipid metabolism
   pathway, phospholipase D signaling pathway, and the
   phosphatidylinositol signaling pathway ([105]Table 4).

Table 4.

   KEGG pathways of the differentially expressed genes (DEGs) and proteins
   (DEPs) related in liver fat metabolism.
   Map ID  Map Name                              DEGs/DEPs
   ko00120 Primary bile acid biosynthesis        CYP27α1
   ko04976 Bile secretion                        ATP1α
   ko00561 Glycerolipid metabolism               LPIN
   ko04072 Phospholipase D signaling pathway     arf1_2/PTK2B
   ko04070 Phosphatidylinositol signaling system plcd/PLCD
   [106]Open in a new tab

   DEGs are represented in italics, while DEPs are represented in regular
   font.

   As shown in [107]Figure 7, the key protein CYP27A1 and the two key
   genes atp1a and plcd were upregulated by dietary taurine intervention
   and were co-enriched in the primary BA synthesis, bile secretion
   pathways, or/and phosphatidylinositol signaling pathway, promoting BA
   synthesis and transport. Meanwhile, the three key proteins LPIN, PLCD,
   and PTK2B and the key gene arf1_2 were downregulated by dietary taurine
   intervention and were co-enriched in the glycerolipid metabolism
   pathway, phospholipase D signaling pathway, or/and phosphatidylinositol
   signaling pathway, resulting in a reduction in TG synthesis. The
   results showed that dietary taurine addition in 15% high-fat diets
   could improve liver fat metabolism of groupers through accelerating BA
   synthesis and transport and inhibiting TG synthesis.

Figure 7.

   [108]Figure 7
   [109]Open in a new tab

   The network diagram of differentially expressed genes and
   differentially expressed proteins. CYP27A1, cholesterol 27-hydroxylase;
   ATP1A, sodium/potassium−transporting ATPase subunit alpha; LIPN,
   phosphatidate phosphatase LPIN; ARF1_2, ADP−ribosylation factor 1/2;
   PTK2B, protein tyrosine kinase 2 beta; PLCD, phosphatidylinositol
   phospholipase C.

4. Discussion

   The results of our present study showed that the growth rate and feed
   utilization of groupers did not differ as the dietary fat level was
   increased from 10% to 15%, which indicates that groupers have a high
   tolerance to dietary fat. Similar results were observed in previous
   studies with aquatic animals [[110]37] such as black sea bream
   [[111]38] and large yellow croaker [[112]39]. However, feeding a 15%
   fat diet led to increased liver fat deposit compared with feeding a 10%
   fat diet in this study. The increase in liver fat deposit was also
   observed in high-fat fed fish in many previous studies
   [[113]8,[114]39,[115]40,[116]41,[117]42,[118]43,[119]44].
   Interestingly, our current study showed a significant effect of
   reducing liver fat, accompanied with improved growth rate and feed
   utilization after intervention with taurine on 15% fat fed groupers, as
   evidenced by previous studies with other fish species such as
   Monopterus albus, California yellowtail, and yellowfin seabream
   [[120]13,[121]18,[122]45]. Concomitantly, the reduced hepatosomatic
   index was also observed in the present study and other previous studies
   after intervention with dietary taurine addition in high-fat fed fish
   such as California yellowtail, yellowfin seabream, white grouper,
   turbot, and rice field eel [[123]18,[124]45,[125]46,[126]47,[127]48].

   Given the aforementioned effects of dietary taurine on reducing liver
   fat in fish, there may be a major regulation of taurine in liver fat
   metabolism at the proteomic level. For this purpose, we conducted
   proteomic analysis for the first time on the liver fat-lowering effect
   of dietary taurine in high-fat fed groupers and the possible molecular
   mechanisms involving fat metabolism. The results showed that a total of
   133 DEPs were identified in the comparison between the 15% fat diet
   group and the 15% fat + 1% taurine group, of which 51 were upregulated
   and 82 proteins were downregulated. The KEGG pathway analysis on these
   DEPs showed that the DEPs were mainly enriched in primary bile acid
   synthesis, glycerophospholipid metabolism, the phosphatidylinositol
   signaling system, and the AMPK signaling pathway, which was found to be
   directly related to liver fat metabolism [[128]49,[129]50], and four
   DEPs (cholesterol 27-hydroxylase, phosphatidate phosphatase LPIN,
   6-phosphofructo-2-kinase, and phosphatidylinositol phospholipase C)
   directly participated in the liver fat metabolism process after dietary
   taurine intervention in high-fat fed groupers. The findings of the DEPs
   and signaling pathways will provide a better understanding of the
   molecular regulation mechanisms of taurine in fat metabolism in fish.

   It is clear that bile acids (BAs) are major components of bile and play
   a vital role in fat metabolism in mammals and fish [[130]39,[131]51].
   They can promote fat transport and absorption in the intestine
   [[132]52]. Taurine-conjugated BAs are the major form of BAs in finfish
   [[133]14,[134]23,[135]53,[136]54]. The reduction in BAs caused by
   dietary taurine addition indicates a reduction in their re-absorption
   in the intestine of finfish [[137]23]. On the other hand, BAs also act
   as ligands to activate farnesoid X receptors (FXRs) [[138]55,[139]56].
   The activated FXRs can reduce lipogenesis and promote fatty acid
   β-oxidation through inhibiting the transcription of sterol regulatory
   element-binding protein 1c and carbohydrate response element-binding
   protein [[140]57]. This is crucial for the regulation of glucose and
   fat homeostasis and cellular inflammatory pathways [[141]57]. The
   synthesis of BAs is regulated by the rate-limiting enzymes CYP7A1 and
   CYP27A1 [[142]51,[143]56], and the resultant taurine-conjugated BAs
   converted from cholesterol are highly hydrophilic, which enhances the
   solubility and excretion of cholesterol [[144]58]. CYP27A1 belongs to a
   family of cytochrome P450 enzymes that converts cholesterol into
   25(R)-26- hydroxycholesterol in the alternative pathway of BA synthesis
   [[145]55,[146]56]. 25(R)-26- hydroxycholesterol can be 7α-hydroxylated
   to 3β, 7α-dihydroxy-5-cholestanoic acid by oxysterol 7alpha-hydroxylase
   for synthesis of chenodeoxycholic acid (CDCA) and cholic acid (CA)
   [[147]56]. CDCA and CA can undergo conjugation with glycine or taurine
   prior to secretion in the bile to form glycocholic acid,
   glycochenodeoxycholic acid, taurocholic acid (TCA), and
   taurochenodeoxycholic acid (TCDCA) [[148]56]. In the present study,
   CYP27A1 expression was upregulated by dietary taurine addition and was
   enriched in the primary BA synthesis pathway ([149]Figure 5), which
   stimulated cholesterol conversion to conjugated BAs in the liver,
   reflecting the hypocholesterolemic effect of taurine [[150]54], thereby
   exhibiting a fat-reducing effect.

   In the present study, the expression of both phosphatidate phosphatase
   LPIN (LPIN) protein and phosphatidylinositol phospholipase C (PLCD)
   protein was downregulated in the 15% fat diet with 1% taurine vs. the
   15% fat diet, and they were enriched in glycerolipid metabolism and the
   phosphatidylinositol signaling system ([151]Figure 5), respectively. In
   the synthesis of neutral TG in animals, glycerol 3-phosphate is
   converted into phosphatidic acid through transacylation, which then
   undergoes dephosphorylation by LPIN to form diacylglycerol (DAG), in
   turn being converted into TG through transacylation [[152]49]. The LPIN
   family acts on the third step in the pathway to form DAG by
   dephosphorylating phosphatidic acid [[153]59]. Lack of LPIN1 expression
   could affect total cholesterol accumulation in mouse adipose tissue
   [[154]60,[155]61]. Moreover, PLCD can hydrolyze the
   phosphatidylinositol 4,5-bisphosphate (PI (4,5) P2) into DAG and
   inositol 1,4,5-trisphosphate [[156]62]. The downregulated PLCD
   prohibits the conversion of PI (4,5) P2 to DAG. Therefore, the
   reduction in TG synthesis in the liver is attributed to the
   downregulation of expression of LPIN and PLCD in the glycerolipid
   metabolism pathway and phosphatidylinositol signaling system.

   The expression of 6-phosphofructo-2-kinase (PFKFB1) was upregulated in
   the 15% fat diet with 1% taurine in comparison with the 15% fat diet,
   and the protein was enriched in the AMPK signaling pathway ([157]Figure
   5) in our current study. PFKFB1 is a bifunctional enzyme that catalyzes
   the synthesis and degradation of fructose 2,6-bisphosphate, acting as
   an allosteric activator of phosphofructokinase-1 and an inhibitor of
   fructose 1,6-bisphosphatase, which confers to PFKFB1 a key role in the
   control of glycolysis and gluconeogenesis [[158]63]. Earlier studies
   have shown that upregulation of PFKFB1 expression is induced by AMPK
   activation [[159]64]. AMPK stimulates catabolism by activating glucose
   uptake, glycolysis (due to PFKFB1 activation), glucose oxidation, and
   fatty acid oxidation [[160]65]. Thus, the upregulation of PFKFB1
   expression in the liver could be triggered by activated AMPK due to
   dietary taurine addition in the present study. The acetyl CoA
   carboxylase (ACC) and fatty acid synthase (FAS) are the key enzymes in
   fat synthesis [[161]1], and were inhibited by activated AMPK in the
   liver of yellow catfish [[162]66]. Therefore, the fat-reducing effect
   of dietary taurine in the liver of groupers could be achieved through
   degradation and utilization of glucose and fat by upregulating PFKFB1
   expression through activating the AMPK signaling pathway in the present
   study.

   To validate the reliability of RNA−Seq data, eight randomly selected
   DEGs in the taurine and high-fat comparison groups were examined using
   qRT−PCR. The fold changes obtained with qRT−PCR were consistent with
   the values obtained with RNA−seq for the six selected genes (arf1_2,
   gk, atp1a, camk, cdo1, and cact), suggesting our RNA-Seq data and the
   results based on RNA−Seq data analysis were reliable [[163]22]. With
   association analysis of transcriptomic and proteomic data through KEGG,
   three DEGs and four DEPs were co-enriched into primary BA synthesis,
   bile secretion, the glycerolipid metabolism pathway, the phospholipid D
   signaling pathway, or/and the phosphatidylinositol signaling system.
   Compared with the 15% fat diet, taurine intervention upregulated the
   expression of the key protein CYP27α1 and the key gene ATP1α. Moreover,
   CYP27α1 and ATP1α were enriched into the primary BA synthesis and bile
   secretion pathways, respectively. The findings indicated that dietary
   taurine intervention helps promote BA synthesis in the liver and
   accelerates BA secretion and transport from the liver. It is well known
   that BAs have a good ability to assist in the digestion of fat in the
   intestine [[164]52,[165]54,[166]58].

   In the meantime, taurine intervention inhibited the expression of the
   key gene arfi_2 and the key proteins LPIN, PLCD, and PTK2B in the liver
   of high-fat fed orange-spotted groupers which were co-enriched into the
   glycerol fat metabolism pathway, the phospholipase D signaling pathway,
   or/and the phosphatidylinositol signaling system pathway. Although the
   associated pathways varied, the metabolites of these pathways directly
   or indirectly point to DAG, which is known to be a precursor substance
   for TG synthesis [[167]49]. In this sense, the reduction in TG
   accumulation in the liver was closely associated with the
   downregulation of key gene and key proteins caused by taurine
   intervention in high-fat fed groupers. In addition, we previously
   reported that the reduction in liver fat deposition caused by dietary
   taurine addition in high-fat fed groupers was the result of the
   decrease in the content of TG molecules at the lipidomic level
   [[168]20]. The increased TG molecules may be achieved by simultaneously
   upregulating the expression of the key gene arfi_2 and the expression
   of the key proteins LPIN and PLCD after taurine intervention. It is
   puzzling that we observed that the 15% fat diet with 1% taurine
   upregulated the expression of the key gene plcd and downregulated the
   expression of the key protein PLCD compared to the 15% fat diet in the
   present study, with inconsistent expression results of the same gene
   and protein. It is precisely because of the complexity of biological
   metabolic regulation that it is necessary for us to conduct in-depth
   analysis of the reasons for the inconsistent results in the future.

5. Conclusions

   In the present study, new DEPs were identified and were enriched into
   metabolic pathways related to liver fat metabolism through proteomic
   analysis and its association with transcriptomic analysis. Three key
   DEGs (atp1_a, arf1_2, and plcd) and four key DEPs (CYP27α1, LPIN, PLCD,
   and PTK2B) were identified and co-enriched into the related pathways in
   liver fat metabolism. The expression changes in these key DEGs and DEPs
   were associated with increased BA biosynthesis and reduced TG
   biosynthesis, thereby mediating the effect of reducing fat in the liver
   after taurine intervention in high-fat fed orange-spotted groupers. To
   our knowledge, this study presents the first proteomic analysis and its
   integration analysis with transcriptomics on the fat-reducing effect of
   dietary taurine in the liver and reveals the close relationship between
   taurine and liver fat metabolism in the fish species. Further study
   will be carried out to investigate the functions and roles of these key
   proteins and key genes as target proteins and genes in the regulation
   of liver fat metabolism in fish in the future.

Author Contributions

   Investigation, methodology, writing-original draft preparation, Y.Z.;
   investigation, data curation, writing-original draft preparation, F.B.;
   data curation, formal analysis, R.X.; formal analysis, visualization,
   M.C.; project administration, writing-review and editing, Y.S.;
   supervision, funding acquisition, writing-review and editing, J.Y. All
   authors have read and agreed to the published version of the
   manuscript.

Institutional Review Board Statement

   Experimental design and procedures in this study were reviewed and
   approved by the Animal Ethics Committee of Jimei University, Xiamen,
   China (Approval number: 2011-58, approval date: 19 December 2011).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The data that support the findings of this study are available from the
   corresponding author upon reasonable request.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This study was supported by funding from the Natural Science Foundation
   of Xiamen City (Grant No. 3502Z202373027) and the National Natural
   Science Foundation of China (Grant No. 32072990).

Footnotes

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References