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Introduction
Recovery of a functional β-cell mass by stimulating pancreatic regeneration is an approach for the treatment of diabetes
that is characterized by absolute or relative deficiency of functional pancreatic
β-cells. However, the capacity of pancreatic β-cells to regenerate in adults is very limited because of terminal differentiation. This notwithstanding, regeneration of
β-cells has indeed been induced in adult animal pancreata by using particular
experimental conditions, such as pancreatectomy (Px), administration of streptozotocin (STZ), wrapping the pancreas in cellophane, or using
transgenic mice with overexpression of γ-interferonin the
β-cells[1]. An understanding of how new
β-cells are generated in these conditions is important with respect to the possibility
of stimulating the regeneration of β-cells in
humans to provide a cure for diabetes.
Many studies have investigated specific markers for the identification of resources such as pancreatic stem or progenitor
cells during β-cell regeneration. A number of putative markers that are transiently expressed in embryonic ducts have been
suggested as indicators of islet stem/progenitor cells, including cytokeratins,
β-galactosidase, PDX-1, tyrosine hydroxylase (TH), and the glucose transporter GLUT
2[1]. However, Dor et al reported that neogenesis
β-cells were formed by duplication of the pre-existing
β-cells rather than differentiation from stem cells in Px
rats[2].
Knowledge about molecular events taking place during pancreatic regeneration would help to identify those molecular
factors regulating cell replication and differentiation during the neogenic renewal of pancreatic tissue. Recently, Rafaeloff
and colleagues reported the expression of islet neogenesis-associated protein (INGAP) in regenerating hamster pancreas
induced by cellophane wrapping, and its expression appeared to stimulate duct cell proliferation, which is a crucial process
in pancreatic neogenesis[3]. Min et
al have reported that clusterin may play essential roles in the neogenic regeneration of
pancreatic tissue by stimulating the proliferation and differentiation of duct
cells[4,5].
These studies on single pathways have thus far been insufficient to fully delineate the complex molecular mechanisms of
pancreatic β-cells regeneration. Although some studies have reported that composite pancreatic extracts from regenerating
pancreas can differentiate rat mesenchymal cells into insulin-producing
cells[6] or cure diabetes induced by streptozotocin in
BALB/c mice and enhance HIT-T15 cell proliferation and insulin
secretion[7], it is still unclear which key factors play a role in
these processes of differentiation and proliferation. Furthermore, they have focused on cell growth and proliferation, but
have ignored other pathological changes such as stress and metabolism in the regenerating pancreas. The proteomic
approach offers a high-throughput technology to study a group of proteins simultaneously, which makes it feasible to study
the differential protein expression profiles relating to particular pathophysiological conditions. In the current study, a
regeneration pancreatic model was induced by 90% Px in rats; the total proteins extracted from the regenerating and
non-regenerating pancreas tissue were used in 2-D gel electrophoresis (2-DE), and the proteins that were differentially expressed
were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS). As a
result, not only several cell growth and proliferation-related proteins, but also energy metabolism, amino acid metabolism and
lipid metabolism-related proteins were found.
Materials and methods
Animal experiments Male specific pathogen-free (SPF) Wistar rats weighing 150-160 g were housed at the SPF animal
facility at the Animal Center of Sun Yat-sen University, and allowed access to standard rat chow and water. Rats were
randomly assigned to two experimental groups
(n=6 per group): Px and sham surgery (Sx). The rats in the Px group were
anesthetized with pentobarbital (50 mg/kg bodyweight, ip) and approximately 90% Px was performed as described by
Bonner-Weir et al[8] with some modifications. Briefly, a midline upper abdominal incision was carried out, and the complete tail
portion of the pancreas, together with the spleen and most of the head of the pancreas, were removed by gentle abrasion with
a small nipper. The major blood vessels were left intact so as not to compromise other organs. The remnant (residual
pancreas) was anatomically well-defined compassion tissue within the common pancreatic duct and the first part of the
duodenum. The rats in the Sx group received Sx that consisted of spleen dissection and separation of the duodenum colon
ligament. On the 3rd day after surgery, rats were anesthetized, and the remnant pancreatic tissue of the Px rats and the
corresponding portion of pancreas of the Sx rats were quickly dissected, cleared on ice and then rapidly stored at -80 °C.
Sample preparation Each sample of tissue (approximately 0.1 g wet weight) was cut into fragments and suspended in 300
µL lysis buffer consisting of 7 mol/L urea, 2 mol/L thiourea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-propanesulfonate
(CHAPS), 20 mmol/L Tris, 5 mmol/L tributyl phosphine (TBP), 0.5% immobilized pH gradient (IPG) buffer [pH 3-10 non linear
(NL)] and 0.6 mmol/L phenylmethane-sulfonyl fluoride (PMSF). The suspension was sequentially homogenized by a rotating
blade homogenizer for 25 s and by sonication 15 times, and then centrifuged at 20
000×g at
4 °C for 30 min. Approximately 280 µL of supernatant was obtained and incubated at room temperature for 1.5 h. After
addition of 20 mmol/L iodoacetamide (IAA), the supernatant was placed away from light and incubated at room temperature
for 1.5 h. A fourfold volume of cold acetone was added slowly and then the supernatant was stored at -20 °C overnight. After
centrifugation at 12 000×g at 4 °C for 15 min, the precipitate was obtained and suspended in 200 µL rehydration buffer [7
mol/L urea, 2 mol/L thiourea, 2% CHAPS,
0.5% IPG buffer (pH 3-10 NL) and 0.002% bromophenol blue] and stored at -80 °C for use. The concentrations of the
extractions were determined by using the 2D Quant kit (Amersham Biosciences, Uppsala, Sweden) according to the
manufacturer¡¯s instructions.
Two-dimensional gel electrophoresis For one-dimensional isoelectric focusing (IEF), 600 µg protein extracted from each
rat was respectively applied to 24 cm immobilized pH 3-10 non linear gradient strips (Amersham Biosciences, Uppsala,
Sweden). IEF was conducted using an IPGPhor II system (Amersham Biosciences, Uppsala, Sweden) according to the
following procedure: 1 h at 200 V, 1 h at 500 V, 1 h at 1000 V then 8-12 h at 8000 V until 80 000 V h. After IEF, the strips were
equilibrated for 25 min with gentle shaking in 10 mL of a solution containing 50 mmol/L Tris-HCl, 6 mol/L urea, 30% glycerol,
2% w/v sodium dodecylsulfate (SDS), and a trace of bromophenol blue. For the second dimensional separation, the IPG strips
were placed on the top of 12% SDS-polyacrylamide gel electrophoresis (PAGE) gels and sealed with 0.5%
w/v agarose in SDS electrophoresis buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.1%
w/v SDS). The molecular weight markers used were the
LMW Calibration Kit for SDS Electrophoresis (Amersham Biosciences, Uppsala, Sweden). Electrophoresis was performed at
10 °C in the Ettan DALTsix system (Amersham Biosciences, Uppsala, Sweden), according to the following program: 2.5 W per
gel for 30 min, then 17 W per gel for 5 h until the bromophenol blue front had migrated to the end of the gel. Gels were stained
with Coomassie blue for 15 min and destained with 10% glacial acetic acid for 12 h. The gels were scanned in Imagescanner
II (Amersham Biosciences, Uppsala, Sweden), and the 2-D images were analyzed with ImageMaster 2D Platinum software
5.0 (Amersham Bio-sciences, Uppsala, Sweden) according to the protocols provided by the manufacturer. To account for
experimental variation, we ran and analyzed 6 gels for each experimental group.
Spot handling Selected protein spots were subjected to fully automated spot handling in the Ettan Spot Handling
Workstation (Amersham Biosciences, Uppsala, Sweden). The methods included spot picking, digestion, extraction of tryptic
peptides, and spotting on Ettan MALDI target slides which were automatically run overnight.
In the automated procedure, gel plugs were cut by a 1.4-
µm picking head, and washed twice in 50% methanol/50 mmol/L
ammonium bicarbonate and once in 75% acetonitrile before drying. For digestion, 10 µL trypsin solution (0.02 µg/mL;
sequencing grade, Promega) was added before incubation at 37 °C for 2 h. Extraction was performed in 2 steps by the addition
of 50% acetonitrile and 0.1% trifluoroacetic acid. The pooled extract was dried and dissolved in 3 µL matrix
(5 mg/mL recrystallized a-cyano-4-hydroxy-cinnamic acid). In the final step before MALDI-ToF (time-of-flight) analysis, 0.3
µL dissolved sample was spotted on the target slides.
Protein identification Peptide mass fingerprinting (PMF) was performed by using an Ettan MALDI-ToF Pro (Amer-sham
Biosciences, Uppsala, Sweden). For each sample, spectra were acquired in the delayed extraction and reflector mode, and an
average of 200 spectra that passed the accepted
criterion of peak intensity were automatically selected and accumulated. Using ProFound data
acquisition[9], spectrum processing and database searches were performed in automatic mode with internal calibration using trypsin autolysis peaks
(m/z 842.509 and m/z 2211.104).
Results
2-D gel separation of proteins To analyze the proteome in relation to
β-cell proliferation and differentiation in rats, a rat
Px-induced pancreatic regeneration model was estab-lished. The proliferating pancreatic tissue derived from rats 3 d after
undergoing Px was processed for 2-D electrophoresis to isolate the candidate proteins that were differentially regulated when
compared with control tissue from Sx rats. In order to measure the reproducibility of the technique, 2-DE for the regenerating
and normal pancreas from the Px and Sx rats was repeated 6 times. For the regenerating pancreatic tissues, a total of 1315±28
spots were detected, and 1098±19 spots were matched with an average matching rate of 83.5%. For the control pancreatic
tissues of the Sx rats, 1369±28 spots were detected in total, and 1110±21 spots were matched, with an average matching rate
of 81.1%. The regeneration 2-DE maps were compared with the
control 2-DE maps, and a total of 997±16 spots were
matched. The patterns of protein expression by the pancreas after Sx and Px are shown in Figure 1. The average volume of
each spot was calculated using 3 gels (selected from the 6 gels in each group), and the ratio of the average volume of each
spot in the Px pancreas relative to the Sx pancreas were determined by using ImageMaster 2D Platinum 5.0 software (Figure
2). Among 91 spots in the gels that had a significant and at least 1.5-fold change in abundance after Px, 41 protein spots were
upregulated and 50 spots were downregulated (selection criteria:
P<0.05 according to Student¡¯s t-test). Figure 1A and 1B
show that differentially expressed proteins (indicated with a circle) were found to have a 1.5-fold or greater difference in
intensity between the regeneration and control groups. Figure 2 shows the images from 2-D-PAGE focusing on some areas
containing differentially expressed proteins. All the 91 differentially expressed proteins were selected for subsequent
analysis by mass spectrometry.
Protein identification All 91 spots of interest were specifically digested by trypsin and 89 spots had nearly perfect
peptide masses for PMF analysis. The PMF maps were
obtained by MALDI-ToF-MS and calibrated against trypsin auto-degraded peaks
(m/z 842.509 and m/z 2211.104). PMF of the
selected spots and a subsequent database search
revealed the identity of these proteins as summarized in Table 1.
The NCBInr database (comprehensive, non-identical protein
database) and a database of homology predicting protein sequences from the rat genome were searched for theoretical
protein digest patterns matching the experimentally determined masses. Figure 3 shows the PMF map of spot #8170, which
database searching revealed was vimentin.
A total of 75.82% (69/91) of selected spots could be identified by MALDI-ToF-MS analysis, whereas some corresponded to
the same protein. For example, spots #8114, #8170, #8174, and #8276 were identified as the same protein, vimentin (Table 1,
Figure 1B). In total, 53 differentially expressed proteins were identified by PMF, including cell proliferation-related proteins,
lipid and energy metabolism-related proteins, protein and amino acid metabolism-related proteins, and signal transduction
and acute-phase response proteins (Table 1).
Discussion
The rat partial pancreatectomy model has been widely used to study diabetes and pancreatic stem cells because it
provides a setting in which the remnant pancreas undergoes regeneration to compensate for the inefficiency of
β-cells. In the present study we attempted to address the molecular basis of
β-cell neogenesis at the tissue level in
vivo, regardless of whether new β-cells are differentiated from stem cells or duplicated from old
β-cells. The strategy used has advantages, and should more accurately reflect the molecular regulation mechanism than cell-level studies
in vitro could, and it should also reveal other important pathological changes accompanying
β-cell neogenesis, which have remained undetected in previous
studies. Our study has identified several proteins whose expression was significantly altered in pancreatectomized rats. In
the following sections, the possible functions of these proteins will be discussed.
Cell growth and proliferation Several proteins that appear to be involved in cell growth, proliferation and related
processes were found to be upregulated in pancreatectomized rats (Table 1). ANXA1 has a significant role in several
physiological and pathological processes, including
anti-inflammation[10], cell growth, differentiation, apoptosis, membrane
fusion, endocytosis and
exocytosis[11-14], and regulation of endocrine
function[15]. ERK2 (one member of the ERK family)
appears to be the major transducer of proliferative signals to the
nucleus[16]. A series of studies provide evidence for Hnrpk
protein involvement in cellular processes such as proliferation and
apoptosis[17-19]. In breast cancer cells, Hnrpk significantly
enhances cell proliferation[18].
Other proteins associated with embryogenesis and cell differentiation have been found to be upregulated in
pancreatectomized rats. L-plastin has been detected in the early
stages of intestinal epithelial cell differentiation until day
14.5, and was localized to the basal surface of the epithelium, but by day 16.5 no
L-plastin was detected in the
epithelium[20]. This result indicates that
L-plastin plays a role during intestinal epithelial cell differentiation. Cytokeratin 8 is the early and fundamental
keratin expressed during development in many
vertebrates[21,22], and is the main keratin present in hyperproliferative human
cells[23,24]. Vimentin is another
intermediate filament (IF) protein, which has already been reported to be rapidly induced during the process of
epithelial-mesenchymal transition and in rapidly proliferating porcine and human pancreatic duct
cells[25,26]. However, mature pancreatic
epithelial cells no longer express vimentin
protein[27]. A study of the hnRNP A2/B1 expression revealed a regulated expression
pattern during fetal development, and down-regulation in normal adult
tissues[28], but re-overexpression occurred during
lung cancer progression[29]. L-arginine: glycine amidinotransferase (AGAT), which is downregulated in pancreatectomized
rats, catalyzes the committed step in creatine biosynthesis. A series of studies found that AGAT fulfiled a function in energy
metabolism while also playing an important role during early embryonic development; during embryogenesis AGAT is
preferentially expressed around the blastopore and later in the notochord of the neurula and tailbud
stages[30].
The potential identification, expansion, and differentiation of adult pancreatic
stem cell(s) raises the possibility of there being enough islets for widespread
β-cell replacement therapy. Recent immunohistochemical observations suggest that
the expression of PDX-1 in pancreatic duct epithelium is upregulated under conditions of pancreatic regeneration in Px rats,
whereas it is transiently expressed in the
embryonic stem cells in foregut endoderm and functionally
directs them toward a pancreas-specific cell fate in
pancreo-genesis[1,31-33]. The authors of these studies considered that the
duct cells expressing the PDX-1 protein transiently
regained their multipotency as progenitor cells in the adult pancreas, and that PDX-1 protein would be one of the markers of
adult pancreatic stem cells. According to this hypo-thesis, these differentially expressed proteins related to
embryogenesis and cell differentiation may be potential markers of pancreatic stem cells. It is exciting that vimentin protein
has been considered as another useful marker for a low-level differentiation stage of pancreatic ductal cells, and one that
correlates with the precursor/progenitor stage during the process of
β-cell neogenesis[34]. Using a proteomic approach, our
own work revealed that other new proteins
(L-plastin, hnRNP A2/B1 and AGAT) shared similar characters with
PDX-1, besides CK8, and vimentin. We postulate that these proteins are associated with
β-cell neogenesis and may be new potential stem cell markers.
Glucose metabolism Four proteins involved in the glycolytic cycle were induced 3 d after Px. Aldolase A catalyzes a
reversible aldol condensation, which cleaves fructose 1,6-bisphosphate to yield two different triose phosphates,
glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Glyceraldehyde 3-phosphate dehydrogenase catalyzes the
phosphate-independent irreversible oxidation of
D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The enzyme
phosphoglycerate mutase catalyzes a reversible conversion of 3-phosphoglycerate to 2-phosphoglycerate.
L-lactate
dehydrogenase (LDH) is the enzyme involved in the final step of anaerobic glycolysis, which catalyzes the interconver-sion
of L-lactate and pyruvate with nicotinamide adenine dinucleotide (NAD+) as a coenzyme.
Several proteins were found to be functionally related to aerobic oxidation, the citric acid cycle and gluconeogenesis:
lipoamide, fumarase and phosphoenolpyruvate carboxy-
kinase were downregulated after pancreatectomy. Lipoamide is a cofactor in the pyruvate
dehydrogenase complex, which catalyzes the oxidative decarboxylation of pyruvate with concomitant formation of
CO2, acetyl-CoA and NADH.
Fumarase catalyzes a reversible hydration of fumarate to
L-malate. Phosphoenolpyruvate carboxykinase plays a central
role in glucose homeostasis as one of the rate-limiting enzymes in gluconeogenesis, which catalyzes the decarboxylation and
mononucleoside triphosphate (NTP)-dependent phosphorylation of oxaloacetate (OAA) to form phosphoenolpyruvate
(PEP) and nucleoside diphosphate (NDP).
Taken together, these data suggest that the pancreas displays substantial anaerobic glycolysis in pancreatectomized
rats; the flux of metabolites from glucose into lactate is enhanced in pancreatic neogenesis. However, the modified
expression of these enzymes may fulfill other biological functions. For example, aldolase A also has an effect on the promotion of
cell growth when overexpressed[35], and GAPDH is a key transcriptional coactivator necessary for entry into S
phase[36].
Lipid metabolism Pancreatic lipase secreted by the exocrine pancreas into the duodenum of the intestine, cleaving
triglycerides into monoglycerides and free fatty acids, was downregulated in Px rats. In addition to pancreatic lipase, the
lipase gene family also encodes other two homologous proteins, pancreatic lipase related proteins 1 and 2 (PLRP1 and
PLRP2). PLRP1 has displayed no significant activity with respect to any of the substrates tested, and its physiological role
is still unknown. Studies on the expression pattern of PLRP1 in rats showed that the mRNA encoding PLRP1 was mainly
expressed shortly after birth and then decreased to a low level as compared with pancreatic
lipase[37]. In the present study, pancreatic lipase and PLRP1 were both downregulated on d 3 after Px, the determination of which ultimately required a study
of sequence expression patterns during pancreatic regeneration.
Three enzymes involved in fatty acid oxidation had decreased expression levels after pancreatectomy. Acyl-CoA
dehydrogenase catalyzes the dehydrogenation of fatty acyl-CoA to
produce a double bond between the a and b carbon atoms
(C-2 and C-3), yielding a trans-D2-enoyl-CoA. Enoyl-CoA
hydratase plays a key role in fatty acid metabolism by catalyzing the
reversible addition of water to
trans-D2-unsaturated enoyl-CoA thioesters.
2,4-Dienoyl-CoA reductase an auxiliary enzyme was needed for
b oxidation of the common unsaturated fatty acids.
It has been shown that the rate of lipid peroxidation is reduced in regenerating pancreas following Px, which accords with
the general hypothesis that increased cell proliferation is associated with a decreased rate of lipid
peroxida-tion[38]. This suggest that the decrease in lipid peroxidation is another important event during pancreatic regeneration. However,
defective triglyceride digestion (pancreatic lipase) and insufficient fatty acid catabolism
would ineffectively generate high-energy metabolites and phospholipids required for cytoplasmic membrane
formation[39]. In this context, modulation of defective lipid
metabolism in pancreatectomized rats might be of therapeutic value.
Amino acid metabolism and protein synthesis
The
expression levels of several enzymes related to amino acid metabolism were downregulated.
2-Amino-3-ketobutyrate-coenzyme A ligase is an enzyme associated with the conversion of
L-threonine to glycine through a 2-step biochemical
pathway[40]. Isovaleryl-CoA dehydrogenase catalyzes the conversion of acyl-CoA thioesters to the corresponding
trans-2-enoyl-CoA, which is involved in leucine
degradation[41]. Branched chain aminotransferases (BCAT) catalyze the transamination of
the branched chain amino acids leucine, isoleucine, and valine to their respective
a-keto acids,
a-ketoisocaproate, a-keto-h-methylvalerate, and
a-ketoiso-valerate[42].
Protein synthesis also seems to be inhibited in the remnants of the pancreas after Px. Several components of the
translational machinery that regulates protein synthesis were observed to be downregulated in our pancreatic regeneration
model. Eukaryotic initiation factor eIF2, which is composed of 3 subunits (a, b, and c), mediates the binding of the initiator
methionyl-tRNA (Met-tRNAi) to the ribosome during the initiation of translation of all cytoplasmic mRNAs in eukaryotic
cells[43]. Eukaryotic elongation factor 2 (eEF2) promotes ribosomal translocation and is involved in eukaryotic polypeptide
chain elongation[44] and some types of post-translational
modification[45-47]. We also found another factor:
high-mobility-group (HMG) protein, a non-histone DNA-binding protein, was downregulated in pancreatectomized rats. HMG box proteins
are generally considered to participate in maintaining the structure of chromatin and to mediate gene expression, replication,
recombination and repair[48].
The effects of amino acid metabolism and protein synthesis on growth and development of the pancreas are largely
unknown. Ip et al reported that pancreatic protein synthesis was transiently low in animals at
birth[49]. Our data suggest that the decrease in protein synthesis of regenerating pancreas was similar with that in pancreatic postnatal development.
The proteome profiling technique used in the present study provided a broad-based and effective approach for the rapid
assimilation and identification of adaptive protein changes during pancreatic regeneration induced by pancreatectomy.
Changes in the expression of proteins that we documented after Px reflect the involvement of various regulation mechanisms:
transcription, translation, post-translation, signal conduction, cell cycle, apoptosis, cellular energy and metabolic pathways,
which would be valuable targets for further investigation. Additionally, information about the dynamic expression
pattern of the regenerating pancreas at different time points after Px would also be very valuable. Recently, Shin
et al investigated differential expression on the 2nd day after 60%
Px[50], which can be added to our data from the 3rd day after 90%
Px to illuminate the molecular mechanism of pancreatic regeneration. In summary, our data elucidate the global proteome
during pancreatic proliferation and differentiation, which is very important and will lead to a better understanding of the
regulation mechanism of pancreatic regeneration, and ultimately assist in reaching the target of discovering protein biomarkers
for pancreatic stem cells.
Acknowledgements
The authors wish to thank Mu-xun ZHANG, Ai-ping ZHANG, and Yan YANG for their assistance with the animal
experiments, Jian-hua ZHANG for assistance with the blood glucose analysis.
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