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Keywords:

  • pancreas;
  • clusterin;
  • regeneration;
  • pancreatectomy;
  • beta-cell

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Based on our previous observations that clusterin induction accompanies pancreas regeneration in the rat, we sought to determine if regeneration might be impaired in mice that lacked clusterin. We studied the impact of absent clusterin on morphogenic and functional features of regenerating pancreas. Clusterin induction was accompanied in the regenerating pancreas by a robust development of new lobules with ductules, acini, and endocrine islets in wild type after partial pancreatectomy. In clusterin knock-out mice, however, pancreatectomy resulted in a poor formation of regenerating lobule. In particular, regeneration of beta-cells was also significantly reduced and was associated with persistent hyperglycemia. Duct cells obtained from pancreatectomized clusterin knock-out mice exhibited impaired beta-cell formation in vitro; this was restored by administration of exogenous clusterin. We suggest that clusterin plays a critical role to promote both exocrine and endocrine regeneration following pancreas injury, as well as for in vitro beta-cell regeneration. Developmental Dynamics 240:605–615, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Pancreatic islet regeneration, either in vivo or in vitro, is an attractive approach for the treatment of type 1 diabetes (Hayashi et al., 2003; Li et al., 2004; Martin-Pagola et al., 2008). While a variety of recent studies have demonstrated the potential for in vitro differentiation of beta-cell phenotypes from embryonic and adult pancreatic stem cells and its potential to be used in islet transplantation (Kroon et al., 2008; Serafimidis et al., 2008), there remains a need to understand critical genes and pathways required for the expansion and differentiation of progenitor cells.

One of the best experimental models of pancreas regeneration involves the surgical removal of 80–90% of pancreatic tissue in young rats (Bonner-Weir et al., 1983, 1993; Min et al., 2003). Partial pancreatectomy (PPx) facilitates regeneration of pancreatic tissues including ducts, acini, and pancreatic islets (Bonner-Weir et al., 1983, 1993; Min et al., 2003; Kim et al., 2004; Joglekar et al., 2007). However, little is known about the molecular regulation of the processes of pancreatic regeneration.

Clusterin is a disulfide-linked heterodimeric glycoprotein expressed ubiquitously in a wide variety of tissues (Laslop et al., 1993; Jordan-Starck et al., 1994; Parczyk et al., 1994; Oda et al., 1994; Min et al., 1998; Shin et al., 2006; Savković et al., 2007). There are two known isoforms of clusterin protein: a secretory form (sCLU) and a nuclear form (nCLU). The sCLU, a predominant form, participates in various physiological and pathological conditions, whereas nCLU acts as a cell death inducer. Due to its multifunctional role, it had been called several names including ApoJ (apolipoprotein J) owing to its action as a lipid transporter (Jones and Jomary, 2002). Although the precise mechanisms of action remain elusive, sCLU may protect cells from protein deposition in extracellular spaces, which is potentially pathological via chaperone action. In terms of a cytoprotective role, its specific interaction with Bax/Ku70 gives rise to sequester Bax from inducing apoptosis (Zhang et al., 2005) and its binding to megalin favors cell survival through Akt activation and Bad phosphorylation, causing a reduction in cytochrome C release (Ammar and Closset, 2008).

Clusterin is an intriguing candidate regulator of pancreatic regeneration. Previously, we reported the expression of pancreatic clusterin during embryonic development and adult pancreas. During development, it is transiently expressed in both alpha and beta-cells (Min et al., 1998). In adult pancreas, clusterin can be detected although weakly in some islet alpha cells, but its expression is considerably up-regulated upon streptozotocin treatment (Kim et al., 2001). We also have shown that transfection-mediated expression of clusterin significantly increases the replication of MIN6 cells and improves the differentiation of beta-cells from rat duct cells (Kim et al., 2006). Moreover, clusterin is specifically expressed in the early process of pancreas regeneration in both developing exocrine and endocrine cells, suggesting a potential role in pancreatic regeneration (Min et al., 2003). These results led us to evaluate endocrine and exocrine pancreas regeneration in clusterin knock-out mice (CLU−/−) during pancreas regeneration after PPx.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Clusterin Expression in the Regenerating Pancreas Following PPx

We assessed clusterin expression in regenerating pancreas of both PPx-CLU−/− and purebred wild type of C57BL/6 mice (PPx-WT) and compared to those of the corresponding sham-operated controls. Clusterin expression was detected in WT pancreas and was significantly increased in developing acini as well as in some islet-forming endocrine cells of the regenerating tissues in pancreatectomized WT mice (PPx-WT) (Fig. 1A,B), in agreement with previous observations (Min et al., 2003), while pancreatectomized CLU CLU−/− mice (PPx-CLU−/−) exhibited complete absence of clusterin expression (Fig. 1A and C).

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Figure 1. Impaired pancreas regeneration in PPx-CLU−/−. A: Clusterin (43 kDa; α-subunit, 64 kDa; whole protein) was detected in the pancreas of WT mice more abundantly after Px. B: Clusterin expression was also evident in histological sections of WT mice. In double-immunolabeled tissue section for clusterin (green fluorescence) and insulin (red fluorescence), clusterin-positive cells were seen in the exocrine tissue (asterisks) as well as in the islet (arrows) with the beta-cells of regenerating pancreas. In contrast, clusterin expression was not detected in both the exocrine and endocrine portions of pancreas (arrows in C) in PPx-CLU−/−. D–I: Defective tissue regeneration was found in PPx-CLU−/−. Six days after PPx, WT mice displayed active tissue regeneration (asterisks in D), whereas PPx-CLU−/− showed significantly defective tissue regeneration (asterisks in E). F: The weight of the regenerating area was also significantly different. G, H: In hematoxylin-and-eosin-stained sections, regenerating pancreas containing developing acini (arrows in G) and small ducts (arrowheads in G and H) was well-demarcated from pre-existing tissue (asterisks in G and H). I: Significant reduction in the regeneration area (dotted line) among whole pancreas remnants was evident in PPx-CLU−/− mice. L, liver; Du, duodenum; Sp, spleen. In G and H, scale bars = 50 μm. In F and I, n = 3, **P < 0.001 versus WT.

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Impaired Exocrine Regeneration in PPx-CLU−/− Mice

In WT mice, we found considerable expansion of the pancreatic rudiments after PPx, indicating active pancreatic regeneration following PPx. In contrast, pancreatic regeneration was impeded after PPx in CLU−/− mice (Figs. 1D,E). The pancreatic remnants taken from PPx-WT was about 1.5-fold heavier compared to those of the PPx-CLU−/− (Fig. 1F). In histological sections, newly formed tissue following PPx was well-demarcated as a separate lobule (regenerating lobule) from the pre-existing pancreas. The regenerating lobule of PPx-WT mice was mainly composed of ductal and acinar structures in various developing stages (Fig. 1G). However, the pancreas of PPx-CLU−/− mice demonstrated impeded development of both ductal and acinar tissues in a swirl of abundant interstitial tissue (Fig. 1H). Morphometric analysis showed a significant reduction of regenerating area in PPx-CLU−/− mice (Fig. 1I). Immunostaining for cytokeratin 20 (CK-20) and amylase can demonstrate regenerating areas in histological sections. We determined the CK-20-positive and amylase-positive areas, respectively. We found that both CK-20-positive and amylase-positive areas were significantly decreased in PPx-CLU−/− mice compared to those of PPx-WT (Fig. 2A–E). We next examined whether the reduced regeneration of pancreatic tissue in PPx-CLU−/− mice could be attributed to reduced pancreatic ductal cell proliferation per se. To evaluate this, we analyzed proliferating cell nuclear antigen (PCNA) in regenerating pancreas by immunohistochemistry. In control animals, PCNA-positive proliferating cells were mostly concentrated in the epithelial linings of ducts. This cell population was significantly reduced in PPx-CLU−/− mice when compared to PPx-WT mice (Fig. 2F). These results suggest that limited regeneration of the pancreatic tissue in PPx-CLU−/− mice could be attributed to insufficient cell proliferation. In addition, we determined proliferative activity of the cell types in the regenerating pancreas by observation of PCNA positivity in the insulin-, CK-20-, and amylase-positive cells. We found only a few proliferating beta cells, whereas many CK-20- and amylase-positive cells were co-labeled with PCNA (Figs. 2G–I).

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Figure 2. Tissue composition and proliferation index in regenerating pancreas of PPx-CLU−/−. Regenerating tissues can be determined in histological sections immunostained for CK-20 (A,B) and amylase (C,D). PPx-CLU−/− mice demonstrated showed a significant reduction of both CK-20 and amylase-positive regenerating tissues determined by microscopic observation (A–D) and morphometric analysis (E) when compared to those of PPx-WT mice. PCNA-positive replicating cells were counted to determine cell proliferation activity in the regenerating pancreas, and a considerable decrease of proliferating cells was shown in the regenerating tissues of PPx-CLU−/− mice when compared to that of PPx-WT (F). In order to determine proliferative activity of cell types in the regenerating pancreas, double labeling of PCNA (red in G–I) with insulin (green in G), cytokeratin-20 (green in H) amylase (green in I) was performed on the regenerating pancreas of PPX-WT mice. Few insulin cells were positive for PCNA (G), whereas numerous CK-20-positive duct cells (arrows in H) and amylase-positive acinar cells were co-labeled with PCNA (arrows in I). Scale bars = 50 μm. E, F: Morphometric analyses data. n = 4, *P < 0.05 versus WT.

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Reduced Beta-Cell Regeneration in the Pancreas in PPx-CLU−/− Mice

We next examined the formation of beta-cells in regenerating lobules at 6 and 12 days after PPx. Insulin-positive cells were associated with pancreatic ducts as single cells or clustered in small numbers (Fig. 3A–D). We also assumed total populations of beta-cells in regenerating lobules (number of insulin-positive cells/mm2) by morphometic analysis and found that growing beta-cells were reduced in PPx-CLU−/− compared to PPx-WT (Fig. 3E), and this was found at the time points of 6 and 12 days after PPx (Fig. 3E) In order to analyze the grade of islet formation, the beta-cells in the regenerating lobules were categorized into four groups. Group I: single cells or small clusters of cells less than 6 beta-cells; group II: beta-cell cluster with 6–10 cells; group III: 11–20 beta-cells as a primitive islets; and group IV: growing islets with over 21 cells. Fewer number of islets were seen throughout the grades, particularly in small-sized islets (group I), when compared to WT mice 6 and 12 days after PPx (Fig. 3F and G). It may indicate that defect of early stage of islet growth in PPx-CLU−/− mice could lead to insufficient islet formation during the period of pancreas regeneration.

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Figure 3. Impaired beta-cell renewal in regenerating pancreas of PPx-CLU−/−. A–D: Histological sections stained for insulin. Variable masses of insulin-positive cells were found including a single cell (arrows in A and B), and islet-like clusters (asterisks in A and B, arrows in C and D). E: Morphometric analysis data showed that a lower number of total beta-cell in PPx-CLU−/− resulted in a substantially lower beta-cell number/mm2 of regenerating area. F,G: The number of insulin-positive cells in regenerating lobules was graded and grouped by counting their numbers. In day 6 and 12, PPx-CLU−/− showed significant reductions in each group when compared to PPx-WT (n=4 each). Black bar, PPx-WT; white bar, PPx-CLU−/−. In A–D, scale bars = 50 μm. *P < 0.05 versus WT.

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Changes in the Expression of Transcription Factors Associated With Exocrine and Endocrine Cell Development

During pancreatic regeneration, the commitment of cells to differentiate into exocrine cells is determined by the expression of several key transcription factors associated with development of pancreatic exocrine tissue (Ptf1a and Hes1) as well as endocrine cell differentiation (pdx-1, isl-1, ngn-3, nkx2.2, and pax4). Quantitative PCR analysis revealed a significant increase of all transcription factors after PPx in WT mice when compared to those of the unoperated controls. In PPx-CLU−/− mice, however, showed no increased expression of transcription factors for exocrine tissue development such as of Ptf1α and Hes1 (Fig. 4A). In the transcription factors for endocrine cell differentiation, up-regulations of the genes, particularly ngn3, nkx2.2, and pax4, were more prominent in both WT and CLU−/− mice after PPx when compared to those of the un-operated WT mice. However, increase of those transcription factors in PPx-CLU−/− was significantly attenuated when compared to those of PPx-WT (Fig. 4B).

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Figure 4. Down-regulation of transcriptional factors associated with pancreatic regeneration in PPx-CLU−/− mRNA expression of transcription factors associated with exocrine (A) and endocrine (B) development. CLU−/− mice showed no increased expression of transcription factors for exocrine tissue development (hes1 and ptf1α), whereas a significant increase was seen after PPx in WT mice (A). The transcription factors for endocrine cell differentiation (ngn3, nkx2.2, and pax4) were considerably increased in WT after PPx, while up-regulations of these genes were significantly attenuated in PPx-CLU−/− (B). The x-fold induction is presented as a relative value to the un-operated WT controls (n = 5). *P < 0.05, **P < 0.001.

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Collectively, these results suggest that insufficient expression of transcription factors associated with exocrine and endocrine differentiation is, at least partly, responsible for impaired pancreatic regeneration in clusterin deficiency.

Alteration of Glucose Metabolism in CLU−/− Mice After PPx

Whereas PPx caused only a moderate increase in blood glucose levels in PPx-WT, fasting blood glucose levels in CLU−/− mice significantly increased 4–12 days after PPx. (Fig. 5A). An intraperitoneal glucose tolerance test performed at day 6 post-PPx also revealed an impaired glucose tolerance in the PPx-CLU−/− (Fig. 5B and C). In addition, tissue insulin content was significantly reduced in pancreas of the PPx-CLU−/− compared to PPx-WT (Fig. 5D). Likewise, serum insulin was also significantly decreased in PPx-CLU−/− mice (Fig. 5E).

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Figure 5. Abnormal glucose homeostasis in PPx-CLU−/−. A: Whereas only a modest increase in fasting glucose level was noted in PPx-WT, PPx-CLU−/− displayed a significantly higher fasting glucose level at day 4 and afterwards. B,C: Intraperitoneal glucose tolerance test performed at day 6 and area under curve (AUC) also revealed a significant difference between the groups. D: Insulin concentrations in pancreas tissue decreased more in PPx-CLU−/− than PPx-WT at day 6 and day 12. E: Similarly, decreased serum insulin levels in PPx-CLU−/− at 6 and 12 days after PPx. n = 5 each, *P < 0.05 versus WT.

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Impaired In Vitro Beta-Cell Differentiation From Ductal Progenitor Cells in CLU−/− Mice

We next examined whether in vitro beta-cell differentiation of the isolated pancreatic ductal cells could be affected by the absence of clusterin. Ductal cells of the PPx-CLU−/− mice displayed poor in vitro differentiation into beta-cells compared to those of the PPx-WT mice (Fig. 6A,B). The differentiation ability of PPx-CLU−/− duct cells was significantly increased following administration of clusterin protein into the culture medium (Fig. 6C). In order to determine the beta-cell nature of the insulin-positive cells during in vitro culture, double immunolabelling for Pdx-1 and MafA was performed and observed by confocal microscope. We found that most of the insulin-positive cells derived from the pancreatic duct of the regenerating pancreas also expressed Pdx-1 and MafA (Figs. 6D–K).

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Figure 6. Defective in vitro beta-cell differentiation of ductal cells isolated from CLU−/− mice. Small fragments of duct tissues were selected under a stereomicroscope and cultured on cover slips. After 3 days of basic culture, cells were subjected to conditional culture with or without indicated concentrations of clusterin. Cells were then incubated with anti-insulin antibody and fluorescein isothiocynate-conjugated secondary antibody and observed by fluorescent microscopy. A, B: Ductal cells isolated from PPx-CLU−/− displayed poor in vitro differentiation into beta-cells. C: The differentiation ability of the PPx-CLU−/− duct cells was significantly increased by clusterin. In order to determine the nature of insulin-positive cells, double immunolabelings for insulin/Pdx-1 (D, E, H, I) and insulin/MafA (F, G, J, K) were performed. Most of the insulin-positive cells (red in D, F, H, and J with DAPI staining in nuclei) were positive for Pdx-1 (E, I) as well as for MafA (G, K). Bars = 50 μm. *P < 0.05 versus WT, **P < 0.05 versus untreated PPx-CLU−/− ductal cells.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In the recent study, we demonstrated that clusterin is an essential protein for the regeneration of pancreatic tissues including insulin-secreting beta-cells. We have used clusterin knockout animals to demonstrate that clusterin plays a powerful role in promoting the entire pancreas regeneration during the post-PPx state. We previously suggested that clusterin is a candidate pancreatic regeneration regulator (Min et al., 2003; Kim et al., 2006).

In the present study, we have demonstrated impaired pancreas regeneration and beta-cell renewal in PPx-CLU−/− mice, implicating clusterin as being pivotal in pancreatic regeneration of both exocrine and endocrine pancreas. Clusterin deficiency impeded various biological processes for tissue regeneration. Absence of clusterin led to lower levels of exocrine cell proliferation, resulting in a reduced mass of regenerating tissue. Insufficient tissue renewal was also evident in endocrine pancreas of CLU−/− mice. As we previously reported in a rat pancreatectomy model (Min et al., 2003), a dynamic morphogenesis of islet-like structure takes place in PPx-WT mice, whereas PPx-CLU−/− mice demonstrated incomplete and insufficient islet formation and beta-cell renewal. Variable sizes of the endocrine cell clusters were evident in the regenerating pancreas, because a single pancreatic endocrine cell originating from the epithelial lining of the duct can migrate into the exocrine parenchyma and gradually aggregate to form a mature islet (Wang et al., 1995; Hayashi et al., 2003). Morphometric data showing a significant reduction in single and small clusters of beta-cells in PPx-CLU−/− mice may account for a failure of early regeneration of islet cells. In agreement with these results, PPx-CLU−/− mice displayed persistent hyperglycemia and abnormal intraperitoneal glucose tolerance test, which were associated with reduced insulin levels in serum and pancreatic tissue. Together, these data imply that impairment of beta-cell regeneration is responsible for abnormal glucose homeostasis in PPx-CLU−/− mice.

The regenerative process of the pancreas is of interest in regard to pathogenesis as well as therapeutic potentials for diabetes. Islet cell regeneration has been repeatedly documented after pancreatic injury in rodent models (Bonner-Weir et al., 1993; Min et al., 2003; Peshavaria et al., 2006). However, it is still not clear whether newly occurring beta-cells are from duplication of pre-existing beta-cells or from differentiation of other cells (Granger and Kushner, 2009; Collombat et al., 2010). Solar et al. (2009) also provided strong evidence that duct cell pluripotency is lost after birth. In contrast, other researchers have provided evidence for the existence of pluripotent stem cells or progenitor cells in adult pancreas (Inada et al., 2008; Xu et al., 2008; Li et al., 2010).

In the present study, we could not find a noticeable increase in the number of self-replicating beta-cells in the regenerating pancreas after PPx, although numerous replicating signals were seen in duct cells as well as in the developing acinar cells. It may be assumed that the new beta-cells derive not only from pre-existing islet cells, but also could develop from duct cells. We speculate that the discrepancy between our data and other studies (Dor et al., 2004) might be caused by the differences in experimental methods. Unlike Dor et al. (2004), we did not inject BrdU during the experiment. Furthermore, we performed 85% PPx by which the amount of remaining tissue must certainly be smaller than that of the previous studies, i.e., 50 or 70% PPx. The difference in the remaining tissue could have modified the manner of pancreas regeneration. In addition, a limited time point of our experiments for detection of beta cell proliferation at 6 days after PPx may be not sufficient to identify proliferating beta cells, since beta cell proliferation and differentiation could occur at different time points. Development of the pancreas requires coordinated interplay among the various transcription factors such as Pdx1, Ngn3, Nkx2.2, Pax4, Isl1, Ptf1a, and Hes1 in the embryonic stage (Ahlgren et al., 1997; Sosa-Pineda et al., 1997; Krapp et al., 1998; Sussel et al., 1998; Gradwohl et al., 2000; Sumazaki et al., 2004). We examined expression levels of these transcription factors in the regenerating pancreas and found that expression levels of major transcription factors were down-regulated in the regenerating pancreas of PPx-CLU−/−. It suggests that clusterin has potential regulatory effects on major transcription factors involved in the development of the pancreas during the regeneration of pancreas.

In vitro beta-cell generation is considered an alternative approach to ensure a sufficient amount of beta-cell surrogates (Sabek et al., 2006; Ouziel-Yahalom et al., 2006; Kayali et al., 2007). In this study, beta-cell regeneration was significantly reduced in duct cells isolated from PPx-CLU−/−, indicative of a critical role of clusterin in vitro transdifferentiation of ducts to beta-cells. In addition, administration of clusterin protein in clusterin-null duct cells significantly increased beta-cell generation. This opens a new possibility that clusterin can be used to increase the mass of beta-cell surrogates in an in vitro system.

In summary, clusterin plays a critical role in the regeneration of both exocrine and endocrine pancreas. Clusterin may provide a critical element in a complex puzzle that could allow for islet regeneration either in vivo or in vitro as part of next-generation diabetic therapeutics.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Clusterin Knock-Out Mice Strains and Animal Care

CLU−/− mice on a background of C57BL/6 (McLaughlin et al., 2000) were maintained in the animal facility of Inha University, College of Medicine, Incheon, Korea. Experimental animals were mated and maintained at 20–23°C with alternating 12-hr cycles of light and dark and provided standard diet and water ad libitum. Newborn mouse pups were littered with their mother until weaning. Fasting blood glucose concentrations were measured using a Mire 3.3G Glucometer (Infopia, Seoul, Korea) in blood obtained from the mouse tail-vein after 5 hr of starvation. Purebred C57BL/6 mice were used as wild type control animals. The mice were used at 12 weeks after birth for PPX, and some neonatal mice were sacrificed in order to examine the development of pancreas.

PPx and Tissue Sampling

Ten-week-old male CLU−/− and WT mice were used for PPx. The animals were fasted for 12 hr before the operation and allowed free access to standard diet and water 5 hr after operation. Approximately 85% of pancreas including gastric and splenic portions was removed by gentle abrasion with a thin wood stick, leaving the major blood vessels supplying other organs intact (see Supp. Fig. S1E, which is available online). The residual pancreas was anatomically well defined as the tissue within 1–2 mm of the common pancreatic duct that extended to the first part of the duodenum. Anesthesia was achieved by inhalation of gaseous nitrous oxide-oxygen and isoflurane. The pancreatic tissues and blood samples were taken from the mice 6 and 12 days after PPx. Sham operations were performed in the same way as PPx without removal of pancreatic tissue. For protein and mRNA analysis, tissue samples were rapidly frozen and stored in liquid nitrogen. The experimental animals were treated humanely and all surgical procedures were performed according to the Animal Use and Care Protocol of Inha University, College of Medicine.

Western Blot Analysis

Pancreatic tissues were taken from mice of PPx-WT and PPx-CLU−/− at 6 days after operation as well as from the sham-operated WT and CLU CLU−/− controls. The pancreatic tissue extracts were homogenized in protein lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, and protease inhibitor cocktail) using a Dounce homogenizer. The protein lysates were centrifuged for 30 min at 12,000 rpm. The clear supernatant was subjected to Western blot. Whole pancreatic protein yield was assessed by absorbance at 280 nm (A280) in lysis buffer using a spectrophotometer. Twenty or thirty micrograms of protein lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immun-Blot™ polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA) in transfer buffer (39 mM glycine, 48 mM Tris base, 0.03% SDS, 20% methanol, pH 8.3) at 80 V for 2.5 hr. Membranes were blocked with 5% non-fat dry milk and 0.1% Tween-20 in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 1 hr. Blots were incubated overnight at 4°C with anti-clusterin and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zymed Laboratories, South San Francisco, CA) for 1 hr. Peroxidase activity was detected by chemiluminescence reaction with WestZol Western blot detection reagent (Intron Biotechnology, Seoul, Korea) according to the manufacturer's protocol.

Isolation and Culture of Pancreatic Duct Cells

Pancreatic ducts were isolated from CLU−/− and WT mice at 6 days after PPx by a modified method for isolation of pancreatic islets (Bouwens et al., 1995; Min et al., 2003). In brief, the whole pancreas was removed from the mouse after laparotomy and injected with 100 μl of collagenase P (50 mg/ml; Roche, Basel, Switzerland) in 5 ml of RPMI 1640 medium (Hyclone, Logan, UT). The tissue fragments were then chopped and digested with the same medium for 20 min at 37°C. The digests were washed with Hanks' buffered salt solution and transferred to a culture dish for rapid sedimentation. After suspension, small fragments of duct tissues 100–150 μm in diameter were selected under a stereomicroscope and plated onto cover slips in 12-well culture dishes for culture. For basic culture, the tissues were cultured with RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin for 3 days. The cultured cells were subjected to conditional culture for 3 days with or without treatment with 10 ng/ml of clusterin protein purified from human serum (Kim et al., 2006). The culture medium was substituted with fresh medium every day during the conditional culture.

Immunocytochemistry

Pancreatic tissue was fixed with Bouin's solution (saturated picric acid: formaldehyde, 3:1) with 0.1% acetic acid (Sigma-Aldrich, St. Louis, MO) for 24 hr at room temperature and embedded in paraffin. Pancreatic sections 4–5 μm in thickness were dewaxed in xylene, and endogenous peroxidase activity was blocked using 0.5% hydrogen peroxide in absolute methanol for 30 min. Sections were then washed in phosphate-buffered saline (PBS) and blocked with normal goat serum or normal horse serum for 30 min at room temperature. The tissue sections were incubated with a 1:200 dilution of goat anti-clusterin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a 1:500 dilution of rabbit anti-insulin (Santa Cruz Biotechnology), a 1:200 dilution of goat anti-cytokeratin 20 (Santa Cruz Biotechnology), a 1:100 dilution of rabbit anti-amylase (Sigma-Aldrich), and a 1:150 dilution of mouse anti-PCNA (Millipore, Billerica, MA). Slides stained for clusterin and CK-20 were incubated with 10 mM citrate buffer for 10 min at 90°C for antigen retrieval before primary antibody application. Slides were incubated with primary antibodies overnight at 4°C before being washed in PBS and incubated with the secondary antibodies (biotinylated anti-mouse, anti-goat, or anti-rabbit IgG, and the avidin-biotin-peroxidase complex; Vector, Burlingame, CA) for 1 hr at room temperature. The reaction was developed using diaminobenzidine tetrahydrochloride (Sigma-Aldrich).

Cultured pancreatic duct cells were fixed with Bouin's solution containing 0.1% acetic acid for 5 min at room temperature. Endogenous peroxidase activity was blocked using absolute methanol containing 0.5% hydrogen peroxide for 5 min at −20°C. For optimal immunolabelling, pancreatic duct cells were incubated with 0.5% triton X-100 in PBS for 5 min and blocked with normal goat serum for 30 min at room temperature. Cultured cells were incubated with a 1:500 dilution of rabbit anti-insulin (1:500, Santa Cruz Biotechnology), anti-PDX-1 (1:500, Chemicon, Temecula, CA), or anti-MafA (Bethyl Laboratories, Montgomery, TX) overnight at °C. For fluorescence staining, secondary antibody was a 1:200 dilution of anti-rabbit FITC (Vector, Burlingame, CA) for 1 hr at room temperature. Stained cells were mounted using VECTASHIELD mounting medium (Vector). Double immune-labeling was performed using pancreatic tissues and cultured cells. In order to avoid the mismatching between the antibodies for double-lableing, two primary antibodies raised from different animal species were applied simultaneously and detected by corresponding secondary antibodies labeled with FITC or TRITC. Double labeling was analyzed using a confocal microscope (Carl Zeiss, Jena, Germany).

Real Time Quantitative PCR Analysis

Real time quantitative PCR was carried out in an iCycler iQ multicolor detection system (Bio-Rad Laboratories) using the pancreatic tissue samples of PPx-WT and PPx-CLU−/− at 6 days after operation as well as from the un-operated WT controls. Reactions were performed in a 20-μl reaction volume using 2×iQ SYBR Green Supermix (Bio-Rad Laboratories). The following primers were used; PTF1a-FWD 5′-catagagaacgaaccaccc tttgag-3′; PTF1a-REV 5′-gcacggagtt tcctggacagagttc-3′, Hes1-FWD 5′-tctac accagcaacagtg-3′; Hes1-REV 5′-tcaaa catctttggcatcac-3′, Ngn3-FWD 5′-tggc actcagcaaacagcga-3′; Ngn3-REW 5′-acccagagccagacaggtct-3′, Isl1-FWD 5′-agatatgggagacatgggcgat-3′; Isl1-REW 5′-acacagcggaaacactcgatg-3′, Pdx-1-FWD 5′-ctcgctgggaacgctggaaca-3′; Pdx-1-REW 5′-gctttggtggatttcatccacgg-3′, Nkx2.2-FWD 5′-cacgcaggtcaagatctg-3′; Nkx2.2-REW 5′-tgcccgcctggaaggtggcg- 3′, Pax-4-FWD 5′-tggctttctgtccttctgtgag g-3′; Pax-4-REW 5′-tccaagactcctgtgcgg tagtag-3′. Amplification was performed by initial polymerase activation for 10 min at 95°C and 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Quantitative values were obtained as threshold PCR cycle number (Ct) when the increase in fluorescent signal of PCR product showed exponential amplification. The target gene mRNA level was normalized to that of 18sRNA in the same sample. In brief, the relative expression level of the target gene compared with that of 18sRNA was calculated as 2−ΔCt, where ΔCt = Ct target gene – Ct (18sRNA). The ratio of relative expression of the target gene was calculated as 2-(ΔΔCt). Each sample was measured in triplicate (Livak et al., 2001).

Intraperitoneal Glucose Tolerance Test (IPGTT)

To examine the normal blood glucose variations, IPGTT was performed in wild type and clusterin deficiency mice. Mice were intraperitoneally injected with 1 mg glucose/g body weight after fasting overnight (14–16 hr). Blood samples were obtained from the tail vein at 0, 5, 10, 15, 30, 60, and 120 min and analyzed for glucose level using Mire 3.3G Glucometer (Infopia Co., Ltd, Gyeonggi-do, Korea).

Measurement of Insulin

Insulin levels in the pancreas and in the serum were determined using a mouse insulin ELISA kit (Mercodia AB, Uppsala, Sweden). Each sample was diluted 1:10 and incubated on mouse monoclonal anti-insulin coated plate. After incubation and washing, bound conjugates were detected by reaction with 3,3′, 5,5′-tetramethylbenzidine. Color changes were analyzed by a spectrophotometric microplate reader. Purified and known concentration calibrators were incubated parallel with unknown samples for standard curves.

Morphometry

To measure the area of regeneration or acinar-rich and duct-rich areas in pancreatic tissue, two paraffin sections showing different tissue profiles in hematoxylin and eosin (H&E) staining were selected from each animal sample. The sections were taken at intervals of approximately 200 paraffin sections. The tissue was photographed with lower magnification (×10) to integrate a whole tissue image using Photoshop program (Adobe Photoshop CS2. Microsoft Co., San Jose, CA) (Supp. Fig. S2). Regenerating pancreas was determined by microscopic observation and indicated on the images. Since the margin of the regenerating lobules was not clearly demarcated due to the abundant fibrous tissue, we lined the outermost acini or ducts to figure out each regenerating lobule in regenerating areas as shown in Figure 1G and H. The dimensions of each regenerating area and total pancreatic area were assessed using scion image program (Scion Corporation, Frederick, MA) (Fig. 1E). The data from 3 different animals in each groups were used for statistical analysis. For standard calculation, each area size (mm2) was converted into percent (%) value. Estimation of duct-rich and acinar-rich areas was similarly performed by regenerating area measurement. Briefly, the two areas were determined by observation of double-stained section with CK-20 and amylase, or two consecutively stained sections, respectively. The areas were determined by the structures demonstrating a predominant immunoreactivity between CK-20 and amylase (Supp. Fig. S3). The areas were indicated on an integrated image of total pancreatic tissue for assessment as described previously.

The number of insulin- or PCNA-positive cells in regenerating lobules was counted and presented as the number of immunoreactive cells/mm2. To determine regenerating activity of the pancreatic islet, total insulin-positive beta-cells and newly developing islets with less than 6 beta-cells were counted in regenerating lobules. For determination of in vitro beta-cell differentiation by immunocytochemistry, we counted the insulin-positive cells of the explants in the immunocytochemically-stained cover-slip (22 × 22 mm), which displayed 40–50 explants. Data on differentiation are presented as the numbers of insulin-positive cells in 1,000 epithelial cells of the explants.

Statistical Analyses

All values are given as the mean ± S.E.M. Differences between two groups were assessed using an unpaired two-tailed t-test. Data from more than two groups were assessed by analysis of variance (ANOVA) followed by a post-hoc least significant difference test. Repeated ANOVA was used for the studies of clusterin knockout and the stage of islet development (i.e., Fig. 3F and G). Statistical analyses were performed using SigmaPlot 10 (Systat Software, Point Richmond, CA).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

This work was supported by the Korea Science and Engineering Foundation by the Ministry of Science and Technology (M10642140004-06N4214-0040) to In-Sun Park.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22556_sm_SuppFig1.tif8317KSupporting Information Figure 1. Pancreas development and impaired expansion of pancreatic tissue at earlier stages of post-PPx period in CLU−/−. Pancreas development was examined at 6 days (A, B) and 12 weeks (C, D) after birth both in WT (A, C) and CLU−/− mice (B, D). Pancreas of young and adult WT and CLU−/− mice showed a similar appearance in size and position (A–D) as well as in weight of adult pancreas (E, n=4). The pancreatic tissues (asterisks in C and D) surrounding splenic vessels indicated by arrows in F and H were removed for PPx from the mice shown in C and D, respectively. Pancreatic tissue surrounding splenic vessels (arrows in) was removed for PPx (F) from the same mice shown in and regeneration was traced 2 days after operation (G). From the CLU−/− mouse shown in D, approximately 85% of pancreatic tissue surrounding splenic vessel (arrows in D, H) was removed by PPx (H) and regeneration was traced 2 days after operation (I). Only a small pancreatic expansion (dotted area in I) was seen in the end of splenic vessel (arrow) in PPx-CLU−/−. In contrast, a prominent expansion of regenerating pancreas (dotted area in G) surrounding splenic vessel (arrow in G) was photographed at 2 days of post-PPx in WT mice (dotted area in G). The area was shown from the same mouse after operation. The histological sections of the pup (J, K) and adult pancreas (L, M) stained with insulin demonstrated normal development of islet beta-cells (arrows in J–M) with abundant exocrine tissues both in WT (J, L) and CLU−/− (K, M) mice.
DVDY_22556_sm_SuppFig2.tif4586KSupporting Information Figure 2. Integrated images of pancreatic tissues for morphometric analysis. The regenerating areas (asterisks in A and B) were discriminated by microscopic observation and marked on the photographs by dotted lines with lower magnification (×10). The percentage of regenerating area can be calculated by an image analyzing system. Regenerating areas of PPx-WT pancreas were larger than that of the PPx-CLU−/−. Scale bars = 200 μm.
DVDY_22556_sm_SuppFig3.tif8088KSupporting Information Figure 3. Determination of duct-rich and acinar-rich areas. The areas were determined by observation of double-stained section with CK-20 (green fluorescence) and amylase (red fluorescence). The areas were determined by immunoreactivity between CK-20 and amylase. A,B: Tissue areas showing predominant CCK-positive ducts are indicated by dotted lines (d), while the areas predominantly composed by amylase-positive cells are identified as an acinar-rich area (a). C: Higher magnification of A. Scale bars = 100 μm.

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