Proteomics alterations in chicken jejunum caused by 24 h fasting

Ethical approval

The experiment followed the European Union principles for animal care and experimentation (EC Directive 86/609/EEC) and it was approved by the local Committee of Animal Welfare, University of Debrecen (registration number: DEMAB/12-7/2015).

Birds and experimental conditions

Experimental setting was the same as described in Simon et al. (2018). Briefly, 24 broiler type (Ross 308 line, all 28-day-old), chickens were allocated to three experimental groups (with eight bird/group), namely ad libitum fed (ad libitum group); fasted for 24 h (F24h group); fasted for 24 h then refed for 2 h (F24hRF2h group).

Intestine morphometric analysis

All chickens were terminated by concussion as recommended in 40/2013. (II. 14.) Hungarian act about animal experiments. Intestinal samples were collected within 10 min after euthanasia. The duodenum (middle part), jejunum and ileum (both one cm from Meckel’s diverticulum) samples (n = 3 per group) were obtained from the three experimental group (ad libitum fed, F24h, F24hRF2h). Intestinal content was flushed out with 1× PBS (2.7 mM KCl, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄, 137 mM NaCl, pH: 7.4) and then fixed in 10% neutral-buffered formalin solution. Tissue sections were stained with hematoxylin and eosin according to Fischer et al. (2008) and digitalized with a Leitz Diaplan microscope (Spot RT-Slider; Diagnostic Instruments, Sterling Heights, USA). Morphometric measures were performed using Photoshop CC 2015 1.2 software (Adobe Systems Inc., San Jose, CA, USA) according to Applegate et al. (1999). First, we measured the scale bar with the ruler tool, then we set a custom measurement scale (510 px = one mm). Finally, the depth of crypts (depth of mucosal invaginations between adjacent villi), the width of muscularis externa and the area and height of villi were recorded (25 technical measurement on each tissue section image).

Protein isolation and fluorescent labeling

Frozen jejunum samples (n = 8, from each group) were crushed to fine powder with mortar and pestle in liquid nitrogen. A total of 50 mg of this powdered sample was weighed with an analytical balance in tubes with low protein affinity (Low-bind; Eppendorf, Hamburg, Germany). Then 500 μl cold lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, pH: 8.8) was added to each one. Samples were placed on ice for 1 h and vortexed at every 10 min (10 s for each). After lysis, homogenates were centrifuged at 16,000×g for 30 min at 4 °C (Centrifuge 5810 R; Eppendorf, Hamburg, Germany). Supernatants were collected to fresh tubes and the protein concentration was determined with RC DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). After the protein precipitation and centrifugation steps the supernatant was decanted and the precipitates were washed twice to ensure detergent compatibility as recommended by the kit. Bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) was used as a concentration standard. Absorbance data were collected with a SPECTROstar Nano (BMG Labtech, Ortenberg, Germany) microplate reader at 650 nm. Sample concentrations were interpolated from a standard curve using MARS data analysis software 3.10 (BMG Labtech, Ortenberg, Germany). The protein concentrations of the lysates ranged between 5.32 and 11.11 μg/μl (Table S1). Supernatants were stored at −70 °C for further use. Prior to the fluorescent labeling pH of the samples were checked with Universal Indicator paper (Rota; VWR, Poole, England) to ensure that all are in the range of pH: 8–9. For each gel 50 μg of sample pool (a mixture of all sample, for normalization) was labeled with 400 pmol of Cyanine2 NHS ester minimal dye (Lumiprobe, Hannover, Germany) and 50 μg from ad libitum and 50 μg from F24h group with each of 400 pmol of Cyanine3 or Cyanine5 using dye swap method (Table 1). Labeling reactions were carried out on ice for 30 min in dark and then 10 mM lysine stop solution was added to each reaction and incubated on ice for 10 min.

Two-dimensional difference gel electrophoresis and image analysis

For the first dimension, seven cm immobilized linear pH gradient strips (pH: 5–8, Bio-Rad, Hercules, CA, USA) were used. We decided to use pH range five to eight as our previous tests showed the majority of proteins in this tissue can be detected in this region. Strips were rehydrated by passive rehydration (using 150 μg of labeled protein) dissolved in 125 μl rehydration buffer (2 M thiourea, 8 M urea, 2% CHAPS, 0.2% (v/v) Bio-Lyte 4/6 and 6/8 ampholyte at a ratio of 1:2, 0.002% (w/v) bromophenol blue) for 16 h at room temperature. Isoelectric focusing was conducted in Protean IEF Cell (Bio-Rad, Hercules, CA, USA). Low voltage (250 V) was applied for 20 min and then, the voltage was gradually increased to 4,000 V over 2.5 h and maintained at that level until a total of 15,349 Vh. Focused IPG strips were equilibrated for 10 min at 50 rpm on orbital shaker (Biosan, Riga, Latvia) in 6 M urea, 20% (v/v) glycerol, 2% (w/v) SDS, 50 mM Tris pH: 8.8 and 2% (w/v) DTT and then for an additional 10 min in the same buffer except that DTT was replaced by 2.5% (w/v) iodoacetamide. After equilibration, proteins were separated by molecular mass using OmniPAGE Mini (Cleaver Scientific, Rugby, UK) vertical electrophoresis system. Electrophoresis was performed on 100 × 100 × 1 mm, 13% polyacrylamide gels (37.5:1 acrylamide:bis-acrylamide ratio) in 1X TGS electrophoresis buffer containing 25 mM Tris, 192 mM glycine and 0.1% (w/v) SDS. The gels were run by applying 90 V until samples entered from the stacking agarose gel (0.5% agarose in 1× TGS) to the separation gel and then 180 V until the tracking dye reached the end of the gels. Preparative polyacrylamide gels for spot picking (containing 500 μg total protein) were stained with colloidal Coomassie G-250 (Thermo Fisher Scientific, Waltham, MA, USA) using a protocol described by Dyballa & Metzger (2009). Gel images were recorded using PharosFX Plus (Bio-Rad, Hercules, CA, USA) scanner with external laser module at 100 μm resolution (254 dpi) using Quantity One 29.0 (Bio-Rad, Hercules, CA, USA) data collector software and exported as 16-bit .tif files (Fig. S1). The images were analyzed with Delta2D 4.3 software (Decodon™ GmbH, Greifswald, Germany). Spots were detected, quantified and normalized according to the volume ratio of corresponding spots detected in the Cy2 image (internal standard) using the in-gel standard warping strategy. Warping is necessary to eliminate the distortions between the gels caused by the run to run differences in electrophoresis in order for proper spot detection. Using the in-gel standard warping strategy, first the sample images (Cy3–Cy5 channels) were distorted to the standard image (Cy2 channel), then all the standard images to a selected standard image.

Protein identification

Spots of interest were cut out from the Coomassie-stained preparative gels and digested according to Shevchenko et al. (2006). Digested samples were prepared to mass spectrometry (MS) in the same way as described by Bhide et al. (2009). Matrix-assisted laser desorption/ionization (MALDI)-MS data were obtained in an automated analysis loop (Ultraflex; Bruker-Daltonics, Leipzig, Germany) equipped with a LIFT-MS/MS device. Spectra were acquired in the positive-ion mode at 50 Hz laser frequency and 100–1,000 individual spectra were averaged. Automated analysis of mass data was performed with FlexAnalysis software (Bruker-Daltonics). MALDI-MS and MALDI-MS/MS data were combined (BioTools; Bruker-Daltonics) to search a non-redundant protein database (Gallus gallus, NCBInr) using Mascot software (Matrix Science, Boston, USA) using the following search parameters: peptide mass fingerprint search, trypsin digestion, unrestricted monoisotopic mass values, 1+ peptide charge state, ± 1.2 peptide mass tolerance and with at maximum one missed cleavages sites.

RNA isolation, reverse transcription, qPCR assays

Whole jejunum samples (eight birds per group) were ground in liquid nitrogen using a mortar and pestle. Powdered tissue (25 ± 2.5 mg) was lysed in TRI Reagent solution (Thermo Fisher Scientific, Waltham, MA, USA), with an Ultra-Turrax T10 at setting five for 1 min. Total RNA was isolated using Direct-zol™ RNA MiniPrep (Zymo Research, Orange, CA, USA) using an on-column DNase I digestion step (using 30 U enzyme for 15 min). Isolated RNA amounts and purity were quantified in eluent using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was checked with 1% agarose gel electrophoresis. Reverse transcription was conducted using 500 ng total RNA with qPCRBIO cDNA Synthesis Kit (PCR Biosystems, London, UK). Reaction conditions were as recommended by the kit’s manual. Prior to qPCR, cDNA samples were diluted 10-fold, then stored in −20 °C until qPCR. Assays were conducted as described by Simon et al. (2018). Reference gene with stable expression (TBP) was revealed from five candidates (Table S2) and used for normalization as a single reference gene as suggested by the reference gene testing method. Details of qPCR primers were specified in Table 2.

Statistical analysis

Statistical analysis of protein spot intensities and qPCR data was performed with GraphPad Prism 7 (La Jolla, CA, USA) software using two-sided, unpaired t-test. Morphometric data was analyzed with one-way ANOVA, followed by Dunn’s post hoc test to compare every mean with each other. Data from qPCR analysis were expressed as group means normalized to mRNA levels of ad libitum fed samples. Results with P < 0.05 were considered as statistically significant.

Small intestine morphology

No significant change was observed between the groups in all measured parameters. One exception was the ileal villus area of the birds that were fasted for 24 h then refed for 2 h. In that case a significant 34% increase was observed compared to the villus area of the birds in the ad libitum fed group (Table S3).

Proteome analysis

After performing the 2D-DIGE experiments, approximately 541 spots were detected on each polyacrylamide gel (Fig. 1). Among them 88 showed statistically significant difference between two experimental groups (ad libitum fed group vs. fasted for 24 h group). After setting a 1.5-fold change cut-off value, seventeen spots showed significant difference in intensity between the two groups (Fig. 2) and sent to MS. Finally, 11 proteins were identified successfully (Table 3).

The identified proteins

Gene expression analysis

All the differentially expressed proteins, identified by MS, were analyzed by quantitative real-time PCR for mRNA expression. Results are shown in Fig. 3. Among the investigated transcripts, MUC6 was showed lower expression, while ACTA2, Keratin, type I cytoskeletal 14 (KRT14), MAGT1, TPM1 and CHP1 showed higher expression (P < 0.05) in the fasted for 24 h group compared to the ad libitum fed group. These results are in accordance with the observed protein level changes (looking at the direction of changes). In case of other genes, no significant difference was detected in their mRNA expression.

Gene expression analysis

All the differentially expressed proteins identified by MS, were also quantified by qPCR at mRNA level. Among them six also showed statistically significant expression, compared to 2D-DIGE. ACTA2 was highly transcribed in the fasted group with similar ratio both at mRNA and protein levels (9.97- and 8.79-fold). The gene MUC6 showed significant decrease in mRNA expression. In case of APOA5, HSP90AA, ACTB, APOA1, EXFABP no significant difference was detected in mRNA expression. However, not all gene showed significant difference compared to protein levels, but the direction of expressional changes are the same as in case of protein level changes.

Morphometric analysis

Morphometric analysis did not found any significant changes in case of jejunum. This may be due to low sample number in our case. However, previous investigation using higher biological replicates clearly showed decrease in villus height and area or crypt number in multiple cases (Yamauchi, Kamisoyama & Isshiki, 1996; Geyra, Uni & Sklan, 2001; Gonzales et al., 2003) after fasting.