To investigate proteins associated with pancreatic regeneration following pancreatectomy, 8-week-old male Sprague-Dawley rats (Daehan Experimental Animals, Seoul, Korea) were anesthetized with isoflurane (Santa Cruz Animal Health^®, Paso Robles, CA, USA) and divided into 3 treatment groups as follows: partial pancreatectomy (n=2; approximately 90% pancreatectomy), sham surgery (n=2) and no surgery (n=2). The animals were allowed free access to standard diet and water before and after the operation. Partial pancreatectomy was executed according to Bonner-Weir et al (32). In brief, 8-week-old male Sprague-Dawley rats (Daehan Experimental Animals) were anesthetized with 3% isoflurane in oxygen (O₂)/nitrous oxide (N₂O) mixtures, and 90% of the pancreatic tissue was then removed by gentle abrasion with cotton swabs, leaving the tissue between the common bile duct and the loop of the duodenum intact. For sham operation, the same surgical procedure was carried out without the removal of pancreatic tissue. On the 3rd day after surgery, the remnant pancreatic tissues were isolated under inhaled anesthesia (3% isoflurane in O₂/N₂O mixture) and the rats were then euthanized. The tissues were fixed with neutral buffered formalin (NBF) for histological examination, and immediately stored at −80°C. The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Konyang University in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals.

Isolated pancreatic tissues were solubilized using a previously described method (33). Solubilized proteins were quantified with a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) and then stored at −80°C until use. For resolution across the pH 4–7 range, 1 mg of protein extracted from the sham-operated and pancreatectomized pancreases was mixed with modified rehydration buffer [6 M urea, 2 M thiourea, 4% CHAPS, 2.5% DTT, 10% isopropanol, 5% glycerol and 2% immobilized pH gradient (IPG) buffer pH 4–7], loaded into a cup on the anodic side of a pH 4–7 IPG strip (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), and incubated for 12 h. Following rehydration, first-dimension IEF was performed on a Multiphor II IEF system (GE Healthcare Bio-Sciences). To separate the second dimension, an equilibrated IPG gel strip was placed on an 8–16% linear gradient SDS polyacrylamide gel and the gels were subjected to electrophoresis using the Ettan-DALT system (GE Healthcare Bio-Sciences). After 2-DE, the gels were stained using colloidal Coomassie brilliant blue (CBB) G-250 (Sigma-Aldrich, St. Louis, MO, USA). The stained gel was scanned with a GS-710 calibrated imaging densitometer (Bio-Rad), and proteins differentially expressed between pancreatectomized and sham operation samples were identified using the Melanie III image analysis software (Swiss Institute for Bioinformatics, Geneva, Switzerland). Two-DE analysis was performed three times independently. Differences among protein spots were analyzed using a two-way Student's t-test.

For the identification of differentially expressed proteins, MALDI-TOF/MS analysis of selected spots was performed as previously described (33). Selected spots sliced from the gel were stained with CBB and then digested with trypsin. Extracted peptides were subjected to MALDI-TOF/MS analysis on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). All acquired spectra of samples were processed using Voyager™ 5.1 software (Applied Biosystems) in the default mode. Averages of 500 spectra were obtained for each sample, and scans were performed twice. Spectra were calibrated automatically upon acquisition using an external 3-point calibration. Peaks were manually assigned using the DATA Explorer™ software package (Applied Biosystems), and spectra were used to search against non-redundant protein sequence databases available online (SWISS-PROT and/or NCBInr Data Bank). The peptide mass fingerprinting data were applied to ProFound and MASCOT search engines (http://prowl.rockefeller.edu/; http://www.matrixscience.com/search_form_select.html) for protein identification based on ProFound and MASCOT scores. The Z score of ProFound is the distance to the population mean in units of the standard deviation.

Pancreatic tissues and cell lines were lysed using RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail (Roche, Basel, Switzerland). To investigate differences in CPB1 phosphorylation, we performed immunoprecipitation. Briefly, 800 µg of lysate was precleared with protein A-agarose for 2 h at 4°C. The supernatant was incubated with anti-CPB1 (GenWay Biotech, San Diego, CA, USA) by shaking overnight at 4°C, followed by incubation with protein A-agarose for 2 h. The beads were resuspended in 2× sample buffer and boiled for 10 min. The proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Immunoblots were incubated in 5% skim milk (Difco, Franklin Lakes, NJ, USA) for 1 h and probed with anti-tyrosine (1:1000; sc-7020; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-CPB1 (1:1000; GWB-31DED0; GenWay Biotech, San Diego, CA, USA) and anti-β-actin (1:3000; sc-47778; Santa Cruz Biotechnology, Inc.) primary antibodies overnight, followed by incubation with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody for 2 h. Immunoreactive bands were detected using an enhanced chemiluminescence kit (ECL; Thermo Scientific, Waltham, MA, USA).

To identify alterations in CPB1 protein phosphorylation levels in the pancreas following pancreatectomy, the sham-operated and partially pancreatectomized pancreases were removed and fixed with NBF overnight at room temperature. Fixed tissues were embedded in paraffin and the paraffin blocks were sliced at 5 µm thicknesses using a tissue microtome (Leica, Nussloch, Germany). The slides were blocked with 10% goat serum (Vector Laboratories, Inc., Burlingame, CA, USA) and incubated with phospho-Tyr antibody (sc-7020; Santa Cruz Biotechnology, Inc.), and CPB1 antibody (GWB-31DED0; GenWay Biotech) as primary antibodies at 4°C overnight and then with Cy2-conjugated goat anti-rabbit IgG (111-225-144) and Cy3-conjugated goat anti-mouse IgG (115-165-146) secondary antibodies (both from Jackson Immuno Research, West Grove, PA, USA) as secondary antibodies at room temperature for 1 h. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole dihydrochloride (Molecular Probes, Waltham, MA, USA). The stained cells were examined under a confocal microscope (Carl Zeiss, Oberkochen, Germany).

Total RNA was isolated from the rat pancreases using TRI Reagent^® (Ambion, Waltham, MA, USA) according to the manufacturer's instructions. Approximately 1.2 kb cDNA of CPB1 (NM_012533.1) was obtained from rat pancreatic RNA by RT-PCR using the following primers: 5′-GCC GCC ACC ATG TTG CTG CTA CTG GCC CT-3′ (forward) and 5′-GGT CCA ATT GGT CAA CAC ACC CA-3′ (reverse), and the cDNA was cloned (CPB1OE) into the EcoRI site of pcDNA3-EGFP (Addgene, Cambridge, MA, USA). Mutations of 6 tyrosine sites of the CPB1 protein (Y298F, Y304F, Y310F, Y312F, Y314F and Y316F) were introduced (CPB1OE-mutY) using the QuikChange^® Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Mutated sites were confirmed by sequencing.

RIN-m insulinoma cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 (ATCC) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland). The cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. The RIN-m cells were transfected with control pcDNA3-EGFP (Addgene), a rat CPB1 full-length cDNA vector (CPB1OE) and mutant rat CPB1 full-length cDNA vector (CPB1OE-mutY) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. To establish stable cell lines, transfected clones were cultured in selective media containing 150 µg/ml G418 (Sigma-Aldrich).

To identify the association between the phosphorylation of CPB1 protein and cell viability, stable cell lines (control, CPB1OE and CPB1OE-mutY) were seeded in 12-well plates at a density of 5×10⁵ cells/well. Cell viability was measured at 6, 24 and 48 h. Cells were collected and mixed with 0.4% trypan blue stain solution (Gibco), after which unstained cells were counted under a microscope (Olympus, Tokyo, Japan).

All graphed data are presented as the means ± standard deviation. The results were analyzed using analysis of variance (ANOVA) or a Student's t-test. P-values of <0.05 and <0.01 were considered to indicate statistically significant and highly statistically significant differences, respectively.

To identify proteins associated with pancreatic regeneration, we compared proteins in pancreatic tissues obtained from rats at 3 days after partial pancreatectomy with those of sham-operated rats by 2-DE analysis with IEF gel electrophoresis on pH 4–7 linear IPG strips. Independent experiments were each carried out in duplicate. Melanie III detected differential intensity in 20 spots; 14 proteins were downregulated and 6 were upregulated in the rats subjected to compared with the sham-operated rats (Fig. 1A and B). The differentially expressed proteins were identified by MALDI-TOF/MS (Table I). Among the identified proteins, phosphoenolpyruvate carboxykinase 2 (PCK2) has been reported to be differentially expressed in pancreatectomy (18). Amylase is also specifically expressed in the pancreas (34). Succinate-CoA ligase ADP-forming β subunit (Sucla2) and isovaleryl-CoA dehydrogenase (Ivd), which are involved in the mitochondrial Krebs cycle, were downregulated upon pancreatectomy. Mitochondrial function and plasticity are associated with glucose circulation (35). Interestingly, carboxypeptidase B (CPB1) precursor protein was identified from 4 spots at different PIs among the 20 spots (Fig. 1C and D). Therefore, we selected CPB1 for further study.

PTMs of proteins such as phosphorylation or dephosphorylation induce shifts in the PI and regulate the functional activity of enzymes (36). Therefore, we investigated alterations in the phosphorylation level of CPB1 in normal, sham-operated and partially pancreatectomized pancreases using the immunoprecipitation method. Phosphorylation of tyrosine residues of the CPB1 protein was significantly reduced by partial pancreatectomy (Fig. 2A). Immunohistochemistry confirmed that the phosphorylation of the tyrosine residue of CPB1 in partial pancreatectomy decreased (Fig. 2B). These results suggested that PTM of the CPB1 protein may be associated with pancreatic regeneration.

An increase in β-cell mass is derived from β-cell neogenesis, proliferation and hypertrophy, and β-cell proliferation gradually declines with age (37). However, partial pancreatectomy models in mice and rats have been reported to enhance β-cell replication (38). Therefore, we investigated whether the PTM of CPB1 was involved in β-cell replication. First, the PTM region of the CPB1 protein was mutated by phenylalanine-to-tyrosine substitution to reduce the phosphorylation level (Fig. 3A). Mutations at 6 sites (Y298F, Y304F, Y310F, Y312F, Y314F and Y316F) were confirmed by sequencing.

Rat β-cell lines overexpressing wild-type or mutant CPB1 were established. CPB1-overexpressing (CPB1OE) cell lines showed higher proliferation than the control (Fig. 3B). In addition, reduced tyrosine phosphorylation in the CPB1OE-mutY cell line resulted in increased proliferation compared to that in CPB1OE cells. To confirm the reduced tyrosine phosphorylation of CPB1, we performed immunoprecipitation of the CPB1 antibody and detected phosphorylated CPB1 by western blotting. Tyrosine phosphorylation of CPB1 in CPB1OE-mutY cell lines decreased (Fig. 3C). Therefore, we clearly demonstrated that the dephosphorylation of CPB1 was associated with pancreatic regeneration.