a SPECIAL ISSUE RESEARCH ARTICLE

Essential Role of Clusterin in PancreasRegenerationSong Lee,1 Seok-Woo Hong,1 Bon-Hong Min,2 Young-Jun Shim,2 Ki-Up Lee,3 In-Kyu Lee,4

M. Bendayan,5 Bruce J. Aronow,6 and In-Sun Park1*

Based on our previous observations that clusterin induction accompanies pancreas regeneration in therat, we sought to determine if regeneration might be impaired in mice that lacked clusterin. We studiedthe impact of absent clusterin on morphogenic and functional features of regenerating pancreas. Clus-terin induction was accompanied in the regenerating pancreas by a robust development of new lobuleswith 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 hyperglyce-mia. Duct cells obtained from pancreatectomized clusterin knock-out mice exhibited impaired beta-cellformation in vitro; this was restored by administration of exogenous clusterin. We suggest that clusterinplays a critical role to promote both exocrine and endocrine regeneration following pancreas injury, aswell as for in vitro beta-cell regeneration. Developmental Dynamics 240:605–615, 2011. VC 2011 Wiley-Liss,Inc.

Key words: pancreas; clusterin; regeneration; pancreatectomy; beta-cell

Accepted 23 December 2010

INTRODUCTION

Pancreatic islet regeneration, eitherin vivo or in vitro, is an attractiveapproach for the treatment of type 1diabetes (Hayashi et al., 2003; Liet al., 2004; Martin-Pagola et al.,2008). While a variety of recent stud-ies have demonstrated the potentialfor in vitro differentiation of beta-cellphenotypes from embryonic and adultpancreatic stem cells and its potentialto be used in islet transplantation(Kroon et al., 2008; Serafimidis et al.,2008), there remains a need to under-

stand critical genes and pathwaysrequired for the expansion and differ-entiation of progenitor cells.

One of the best experimental mod-

els of pancreas regeneration involves

the surgical removal of 80–90% of

pancreatic tissue in young rats (Bon-

ner-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 het-

erodimeric glycoprotein expressed

ubiquitously in a wide variety of tis-

sues (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; Savkovic

et al., 2007). There are two known iso-

forms of clusterin protein: a secretory

form (sCLU) and a nuclear form

(nCLU). The sCLU, a predominant

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Additional Supporting Information may be found in the online version of this article.1Department of Anatomy and Center for Advanced Medical Education, College of Medicine, Inha University, Incheon, Korea2Department of Pharmacology and BK21 Program for Medical Sciences, College of Medicine, Korea University, Seoul, Korea3Department of Internal Medicine, University of Ulsan, College of Medicine, Seoul, Korea4Department of Internal Medicine and Biochemistry and Cell Biology, Kyungpook National University School of Medicine, Daegu, Korea5Department of Pathology and Cell Biology, University of Montreal, Montreal, Quebec, Canada6Division of Molecular and Developmental Biology, Department of Pediatrics, University of Cincinnati, Children’s Hospital Medical Center,Cincinnati, OhioGrant sponsor: Korea Science and Engineering Foundation by the Ministry of Science and Technology; Grant number: M10642140004-06N4214-0040.*Correspondence to: In-Sun Park, Department of Anatomy and BK21 Center for Advanced Medical Education, School ofMedicine, Inha University, Shinheung-Dong, Jung-Gu, Incheon, 400-103, Korea. E-mail: sunpark@inha.ac.kr

DOI 10.1002/dvdy.22556Published online 2 February 2011 in Wiley Online Library (wileyonlinelibrary.com).

DEVELOPMENTAL DYNAMICS 240:605–615, 2011

VC 2011 Wiley-Liss, Inc.

Fig. 1.

Fig. 2.

form, participates in various physio-

logical and pathological conditions,

whereas nCLU acts as a cell death in-

ducer. Due to its multifunctional role,

it had been called several names

including ApoJ (apolipoprotein J)

owing to its action as a lipid trans-

porter (Jones and Jomary, 2002).

Although the precise mechanisms of

action remain elusive, sCLU may pro-

tect cells from protein deposition in

extracellular spaces, which is poten-

tially 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 acti-

vation 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 expres-sion of pancreatic clusterin duringembryonic development and adultpancreas. During development, it istransiently expressed in both alphaand beta-cells (Min et al., 1998). Inadult pancreas, clusterin can bedetected although weakly in some is-

let alpha cells, but its expression isconsiderably up-regulated upon strep-tozotocin treatment (Kim et al., 2001).We also have shown that transfection-mediated expression of clusterin sig-nificantly increases the replication ofMIN6 cells and improves the differen-tiation of beta-cells from rat duct cells(Kim et al., 2006). Moreover, clusterinis specifically expressed in the earlyprocess of pancreas regeneration inboth developing exocrine and endo-crine cells, suggesting a potential rolein pancreatic regeneration (Min et al.,2003). These results led us to evaluateendocrine and exocrine pancreasregeneration in clusterin knock-outmice (CLU�/�) during pancreas regen-eration after PPx.

RESULTS

Clusterin Expression in the

Regenerating Pancreas

Following PPx

We assessed clusterin expression inregenerating pancreas of both PPx-CLU�/� and purebred wild type ofC57BL/6 mice (PPx-WT) and comparedto those of the corresponding sham-operated controls. Clusterin expressionwas detected in WT pancreas and was

significantly increased in developingacini as well as in some islet-formingendocrine cells of the regenerating tis-sues in pancreatectomized WT mice(PPx-WT) (Fig. 1A,B), in agreementwith previous observations (Min et al.,2003), while pancreatectomized CLUCLU�/� mice (PPx-CLU�/�) exhibitedcomplete absence of clusterin expres-sion (Fig. 1A and C).

Impaired Exocrine

Regeneration in

PPx-CLU2/2 Mice

In WT mice, we found considerableexpansion of the pancreatic rudi-ments after PPx, indicating activepancreatic regeneration followingPPx. In contrast, pancreatic regenera-tion was impeded after PPx in CLU�/�

mice (Figs. 1D,E). The pancreatic rem-nants taken from PPx-WT was about1.5-fold heavier compared to those ofthe PPx-CLU�/� (Fig. 1F). In histologi-cal sections, newly formed tissue fol-lowing PPx was well-demarcated as aseparate lobule (regenerating lobule)from the pre-existing pancreas. Theregenerating lobule of PPx-WT micewas mainly composed of ductal and ac-inar structures in various developingstages (Fig. 1G). However, the pan-creas of PPx-CLU�/� mice demon-strated impeded development of bothductal and acinar tissues in a swirl ofabundant interstitial tissue (Fig. 1H).Morphometric analysis showed a sig-nificant reduction of regenerating areain PPx-CLU�/� mice (Fig. 1I). Immu-nostaining for cytokeratin 20 (CK-20)and amylase can demonstrate regener-ating areas in histological sections. Wedetermined the CK-20-positive andamylase-positive areas, respectively.We found that both CK-20-positiveand amylase-positive areas were sig-nificantly decreased in PPx-CLU�/�

mice compared to those of PPx-WT(Fig. 2A–E). We next examinedwhether the reduced regeneration ofpancreatic tissue in PPx-CLU�/� micecould be attributed to reduced pancre-atic ductal cell proliferation per se. Toevaluate this, we analyzed proliferat-ing cell nuclear antigen (PCNA) inregenerating pancreas by immunohis-tochemistry. In control animals,PCNA-positive proliferating cells weremostly concentrated in the epithelial

Fig. 1. Impaired pancreas regeneration in PPx-CLU�/�. A: Clusterin (43 kDa; a-subunit, 64kDa; 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-immunola-beled 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 thebeta-cells of regenerating pancreas. In contrast, clusterin expression was not detected in boththe exocrine and endocrine portions of pancreas (arrows in C) in PPx-CLU�/�. D–I: Defective tis-sue regeneration was found in PPx-CLU�/�. Six days after PPx, WT mice displayed active tissueregeneration (asterisks in D), whereas PPx-CLU�/� showed significantly defective tissue regener-ation (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-existingtissue (asterisks in G and H). I: Significant reduction in the regeneration area (dotted line) amongwhole pancreas remnants was evident in PPx-CLU�/� mice. L, liver; Du, duodenum; Sp, spleen.In G and H, scale bars ¼ 50 mm. In F and I, n ¼ 3, **P < 0.001 versus WT.

Fig. 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) andmorphometric analysis (E) when compared to those of PPx-WT mice. PCNA-positive replicatingcells were counted to determine cell proliferation activity in the regenerating pancreas, and aconsiderable 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 celltypes in the regenerating pancreas, double labeling of PCNA (red in G–I) with insulin (green inG), cytokeratin-20 (green in H) amylase (green in I) was performed on the regenerating pancreasof 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 ¼ 50 mm. E, F: Morphometric analyses data. n ¼ 4, *P < 0.05 versusWT.

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CLUSTERIN AND PANCREAS REGENERATION 607

linings of ducts. This cell populationwas significantly reduced in PPx-CLU�/� mice when compared to PPx-WT mice (Fig. 2F). These results sug-gest that limited regeneration of thepancreatic tissue in PPx-CLU�/� micecould be attributed to insufficient cellproliferation. In addition, we deter-mined proliferative activity of the celltypes in the regenerating pancreas byobservation of PCNA positivity in theinsulin-, CK-20-, and amylase-positivecells. We found only a few proliferatingbeta cells, whereas many CK-20- andamylase-positive cells were co-labeledwith PCNA (Figs. 2G–I).

Reduced Beta-Cell

Regeneration in the Pancreas

in PPx-CLU2/2 Mice

We next examined the formation ofbeta-cells in regenerating lobules at 6and 12 days after PPx. Insulin-posi-tive cells were associated with pancre-atic ducts as single cells or clusteredin small numbers (Fig. 3A–D). Wealso assumed total populations ofbeta-cells in regenerating lobules(number of insulin-positive cells/mm2)by morphometic analysis and foundthat growing beta-cells were reducedin PPx-CLU�/� compared to PPx-WT

(Fig. 3E), and this was found at thetime points of 6 and 12 days after PPx(Fig. 3E) In order to analyze the gradeof islet formation, the beta-cells in theregenerating lobules were categorizedinto four groups. Group I: single cellsor small clusters of cells less than 6beta-cells; group II: beta-cell clusterwith 6–10 cells; group III: 11–20 beta-cells as a primitive islets; and groupIV: growing islets with over 21 cells.Fewer number of islets were seenthroughout the grades, particularly insmall-sized islets (group I), when com-pared to WT mice 6 and 12 days afterPPx (Fig. 3F and G). It may indicate

Fig. 3. Impaired beta-cell renewal in regenerating pancreas of PPx-CLU�/�. A–D: Histological sections stained for insulin. Variable masses of in-sulin-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: Mor-phometric analysis data showed that a lower number of total beta-cell in PPx-CLU�/� resulted in a substantially lower beta-cell number/mm2 ofregenerating area. F,G: The number of insulin-positive cells in regenerating lobules was graded and grouped by counting their numbers. In day 6and 12, PPx-CLU�/� showed significant reductions in each group when compared to PPx-WT (n¼4 each). Black bar, PPx-WT; white bar, PPx-CLU�/�. In A–D, scale bars ¼ 50 mm. *P < 0.05 versus WT.

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608 LEE ET AL.

that defect of early stage of isletgrowth in PPx-CLU�/� mice couldlead to insufficient islet formationduring the period of pancreasregeneration.

Changes in the Expression of

Transcription Factors

Associated With Exocrine and

Endocrine Cell Development

During pancreatic regeneration, thecommitment of cells to differentiateinto exocrine cells is determined bythe expression of several key tran-scription factors associated with de-velopment of pancreatic exocrine tis-sue (Ptf1a and Hes1) as well asendocrine cell differentiation (pdx-1,isl-1, ngn-3, nkx2.2, and pax4). Quan-titative PCR analysis revealed a sig-nificant increase of all transcriptionfactors after PPx in WT mice whencompared to those of the unoperatedcontrols. In PPx-CLU�/� mice, how-ever, showed no increased expressionof transcription factors for exocrinetissue development such as of Ptf1aand Hes1 (Fig. 4A). In the transcrip-tion factors for endocrine cell differen-tiation, up-regulations of the genes,particularly ngn3, nkx2.2, and pax4,

were more prominent in both WT andCLU�/� mice after PPx when com-pared to those of the un-operated WTmice. However, increase of those tran-scription factors in PPx-CLU�/� wassignificantly attenuated when com-pared to those of PPx-WT (Fig. 4B).

Collectively, these results suggestthat insufficient expression of tran-scription factors associated with exo-crine and endocrine differentiation is,at least partly, responsible forimpaired pancreatic regeneration inclusterin deficiency.

Alteration of Glucose

Metabolism in CLU2/2 Mice

After PPx

Whereas PPx caused only a moderateincrease in blood glucose levels inPPx-WT, fasting blood glucose levelsin CLU�/� mice significantlyincreased 4–12 days after PPx. (Fig.5A). An intraperitoneal glucose toler-ance test performed at day 6 post-PPxalso revealed an impaired glucose tol-erance in the PPx-CLU�/� (Fig. 5Band C). In addition, tissue insulin con-tent was significantly reduced in pan-creas of the PPx-CLU�/� compared toPPx-WT (Fig. 5D). Likewise, serum

insulin was also significantlydecreased in PPx-CLU�/� mice (Fig.5E).

Impaired In Vitro Beta-Cell

Differentiation From Ductal

Progenitor Cells in CLU2/2

Mice

We next examined whether in vitrobeta-cell differentiation of the isolatedpancreatic ductal cells could beaffected by the absence of clusterin.Ductal cells of the PPx-CLU�/� micedisplayed poor in vitro differentiationinto beta-cells compared to those ofthe PPx-WT mice (Fig. 6A,B). The dif-ferentiation ability of PPx-CLU�/�

duct cells was significantly increasedfollowing administration of clusterinprotein into the culture medium (Fig.6C). In order to determine the beta-cell nature of the insulin-positive cellsduring in vitro culture, double immu-nolabelling for Pdx-1 and MafA wasperformed and observed by confocalmicroscope. We found that most of theinsulin-positive cells derived from thepancreatic duct of the regeneratingpancreas also expressed Pdx-1 andMafA (Figs. 6D–K).

Fig. 4. Down-regulation of transcriptional factors associated with pancreatic regeneration in PPx-CLU�/� mRNA expression of transcription fac-tors associated with exocrine (A) and endocrine (B) development. CLU�/� mice showed no increased expression of transcription factors for exo-crine tissue development (hes1 and ptf1a), whereas a significant increase was seen after PPx in WT mice (A). The transcription factors forendocrine cell differentiation (ngn3, nkx2.2, and pax4) were considerably increased in WT after PPx, while up-regulations of these genes were sig-nificantly attenuated in PPx-CLU�/� (B). The x-fold induction is presented as a relative value to the un-operated WT controls (n ¼ 5). *P < 0.05,**P < 0.001.

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CLUSTERIN AND PANCREAS REGENERATION 609

DISCUSSION

In the recent study, we demonstratedthat clusterin is an essential proteinfor the regeneration of pancreatic tis-sues including insulin-secreting beta-cells. We have used clusterin knock-out animals to demonstrate that clus-terin plays a powerful role in promot-ing the entire pancreas regenerationduring the post-PPx state. We previ-ously suggested that clusterin is acandidate pancreatic regenerationregulator (Min et al., 2003; Kim et al.,2006).

In the present study, we have dem-onstrated impaired pancreas regener-ation and beta-cell renewal in PPx-CLU�/� mice, implicating clusterinas being pivotal in pancreatic regen-eration of both exocrine and endo-crine pancreas. Clusterin deficiency

impeded various biological processesfor tissue regeneration. Absence ofclusterin led to lower levels of exo-crine cell proliferation, resulting in areduced mass of regenerating tissue.Insufficient tissue renewal was alsoevident in endocrine pancreas ofCLU�/� mice. As we previouslyreported in a rat pancreatectomymodel (Min et al., 2003), a dynamicmorphogenesis of islet-like structuretakes place in PPx-WT mice, whereasPPx-CLU�/� mice demonstratedincomplete and insufficient islet for-mation and beta-cell renewal. Vari-able sizes of the endocrine cell clus-ters were evident in the regeneratingpancreas, because a single pancreaticendocrine cell originating from theepithelial lining of the duct canmigrate into the exocrine parenchyma

and gradually aggregate to form amature islet (Wang et al., 1995; Haya-shi et al., 2003). Morphometric datashowing a significant reduction in sin-gle and small clusters of beta-cells inPPx-CLU�/� mice may account for afailure of early regeneration of isletcells. In agreement with these results,PPx-CLU�/� mice displayed persistenthyperglycemia and abnormal intra-peritoneal glucose tolerance test,which were associated with reducedinsulin levels in serum and pancreatictissue. Together, these data imply thatimpairment of beta-cell regenerationis responsible for abnormal glucosehomeostasis in PPx-CLU�/� mice.The regenerative process of the

pancreas is of interest in regard topathogenesis as well as therapeuticpotentials for diabetes. Islet cell

Fig. 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 day6 and area under curve (AUC) also revealed a significant difference between the groups. D: Insulin concentrations in pancreas tissue decreasedmore 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. n¼ 5 each, *P < 0.05 versus WT.

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regeneration has been repeatedlydocumented after pancreatic injury inrodent models (Bonner-Weir et al.,1993; Min et al., 2003; Peshavariaet al., 2006). However, it is still notclear whether newly occurring beta-cells are from duplication of pre-exist-ing beta-cells or from differentiationof other cells (Granger and Kushner,2009; Collombat et al., 2010). Solaret al. (2009) also provided strong evi-dence that duct cell pluripotency islost after birth. In contrast, otherresearchers have provided evidencefor the existence of pluripotent stemcells or progenitor cells in adult pan-creas (Inada et al., 2008; Xu et al.,2008; Li et al., 2010).

In the present study, we could notfind a noticeable increase in the num-ber of self-replicating beta-cells in theregenerating pancreas after PPx,although numerous replicating sig-nals were seen in duct cells as well asin the developing acinar cells. It maybe assumed that the new beta-cellsderive not only from pre-existing isletcells, but also could develop from ductcells. We speculate that the discrep-ancy between our data and otherstudies (Dor et al., 2004) might becaused by the differences in experi-mental methods. Unlike Dor et al.(2004), we did not inject BrdU duringthe experiment. Furthermore, we per-formed 85% PPx by which the amount

of remaining tissue must certainly besmaller than that of the previousstudies, i.e., 50 or 70% PPx. The dif-ference in the remaining tissue couldhave modified the manner of pancreasregeneration. In addition, a limitedtime point of our experiments fordetection of beta cell proliferation at 6days after PPx may be not sufficientto identify proliferating beta cells,since beta cell proliferation and differ-entiation could occur at different timepoints. Development of the pancreasrequires coordinated interplay amongthe various transcription factors suchas Pdx1, Ngn3, Nkx2.2, Pax4, Isl1,Ptf1a, and Hes1 in the embryonicstage (Ahlgren et al., 1997; Sosa-

Fig. 6. Defective in vitro beta-cell differentiation of ductal cells isolated from CLU�/� mice. Small fragments of duct tissues were selected undera stereomicroscope and cultured on cover slips. After 3 days of basic culture, cells were subjected to conditional culture with or without indicatedconcentrations of clusterin. Cells were then incubated with anti-insulin antibody and fluorescein isothiocynate-conjugated secondary antibody andobserved by fluorescent microscopy. A, B: Ductal cells isolated from PPx-CLU�/� displayed poor in vitro differentiation into beta-cells. C: The dif-ferentiation 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 ¼ 50 mm. *P < 0.05 versus WT, **P < 0.05 versusuntreated PPx-CLU�/� ductal cells.

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CLUSTERIN AND PANCREAS REGENERATION 611

Pineda et al., 1997; Krapp et al.,1998; Sussel et al., 1998; Gradwohlet al., 2000; Sumazaki et al., 2004).We examined expression levels ofthese transcription factors in theregenerating pancreas and found thatexpression levels of major transcrip-tion factors were down-regulated inthe regenerating pancreas of PPx-CLU�/�. It suggests that clusterin haspotential regulatory effects on majortranscription factors involved in thedevelopment of the pancreas duringthe regeneration of pancreas.

In vitro beta-cell generation is con-sidered an alternative approach toensure a sufficient amount of beta-cellsurrogates (Sabek et al., 2006; Ouziel-Yahalom et al., 2006; Kayali et al.,2007). In this study, beta-cell regener-ation was significantly reduced induct cells isolated from PPx-CLU�/�,indicative of a critical role of clusterinin vitro transdifferentiation of ductsto beta-cells. In addition, administra-tion of clusterin protein in clusterin-null duct cells significantly increasedbeta-cell generation. This opens anew possibility that clusterin can beused to increase the mass of beta-cellsurrogates in an in vitro system.

In summary, clusterin plays a criti-cal role in the regeneration of bothexocrine and endocrine pancreas.Clusterin may provide a critical ele-ment in a complex puzzle that couldallow for islet regeneration either invivo or in vitro as part of next-genera-tion diabetic therapeutics.

EXPERIMENTAL

PROCEDURES

Clusterin Knock-Out Mice

Strains and Animal Care

CLU�/� mice on a background ofC57BL/6 (McLaughlin et al., 2000)were maintained in the animal facil-ity of Inha University, College of Med-icine, Incheon, Korea. Experimentalanimals were mated and maintainedat 20–23�C with alternating 12-hrcycles of light and dark and providedstandard diet and water ad libitum.Newborn mouse pups were litteredwith their mother until weaning.Fasting blood glucose concentrationswere measured using a Mire 3.3GGlucometer (Infopia, Seoul, Korea) inblood obtained from the mouse tail-

vein after 5 hr of starvation. PurebredC57BL/6 mice were used as wild typecontrol animals. The mice were usedat 12 weeks after birth for PPX, andsome neonatal mice were sacrificed inorder to examine the development ofpancreas.

PPx and Tissue Sampling

Ten-week-old male CLU�/� and WTmice were used for PPx. The animalswere fasted for 12 hr before the opera-tion and allowed free access to stand-ard diet and water 5 hr after opera-tion. Approximately 85% of pancreasincluding gastric and splenic portionswas removed by gentle abrasion witha thin wood stick, leaving the majorblood vessels supplying other organsintact (see Supp. Fig. S1E, which isavailable online). The residual pan-creas was anatomically well definedas the tissue within 1–2 mm of thecommon pancreatic duct thatextended to the first part of the duo-denum. Anesthesia was achieved byinhalation of gaseous nitrous oxide-oxygen and isoflurane. The pancreatictissues and blood samples were takenfrom the mice 6 and 12 days afterPPx. Sham operations were per-formed in the same way as PPx with-out removal of pancreatic tissue. Forprotein and mRNA analysis, tissuesamples were rapidly frozen andstored in liquid nitrogen. The experi-mental animals were treatedhumanely and all surgical procedureswere performed according to the Ani-mal Use and Care Protocol of InhaUniversity, College of Medicine.

Western Blot Analysis

Pancreatic tissues were taken frommice of PPx-WT and PPx-CLU�/� at 6days after operation as well as fromthe sham-operated WT and CLUCLU�/� controls. The pancreatic tis-sue extracts were homogenized inprotein lysis buffer (50 mM Tris-HCl,pH 8.0, 150 mM NaCl, 5 mM EDTA,1% NP-40, and protease inhibitorcocktail) using a Dounce homogenizer.The protein lysates were centrifugedfor 30 min at 12,000 rpm. The clearsupernatant was subjected to Westernblot. Whole pancreatic protein yieldwas assessed by absorbance at 280nm (A280) in lysis buffer using a spec-

trophotometer. Twenty or thirtymicrograms of protein lysate was sep-arated by 10% sodium dodecyl sul-fate-polyacrylamide gel electrophore-sis (SDS-PAGE) and transferred ontoImmun-BlotTM polyvinylidene fluo-ride membrane (Bio-Rad Laborato-ries, Hercules, CA) in transfer buffer(39 mM glycine, 48 mM Tris base,0.03% SDS, 20% methanol, pH 8.3) at80 V for 2.5 hr. Membranes wereblocked with 5% non-fat dry milk and0.1% Tween-20 in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 1 hr.Blots were incubated overnight at 4�Cwith anti-clusterin and anti-actin(Santa Cruz Biotechnology, SantaCruz, CA) antibodies followed by incu-bation with horseradish peroxidase(HRP)-conjugated secondary antibod-ies (Zymed Laboratories, South SanFrancisco, CA) for 1 hr. Peroxidase ac-tivity was detected by chemilumines-cence reaction with WestZol Westernblot detection reagent (Intron Bio-technology, Seoul, Korea) according tothe manufacturer’s protocol.

Isolation and Culture of

Pancreatic Duct Cells

Pancreatic ducts were isolated fromCLU�/� and WT mice at 6 days afterPPx by a modified method for isola-tion of pancreatic islets (Bouwenset al., 1995; Min et al., 2003). In brief,the whole pancreas was removed fromthe mouse after laparotomy andinjected with 100 ml of collagenase P(50 mg/ml; Roche, Basel, Switzerland)in 5 ml of RPMI 1640 medium(Hyclone, Logan, UT). The tissuefragments were then chopped anddigested with the same medium for 20min at 37�C. The digests were washedwith Hanks’ buffered salt solutionand transferred to a culture dish forrapid sedimentation. After suspen-sion, small fragments of duct tissues100–150 mm in diameter wereselected under a stereomicroscopeand plated onto cover slips in 12-wellculture dishes for culture. For basicculture, the tissues were culturedwith RPMI 1640 medium containing10% fetal bovine serum and 1% peni-cillin/streptomycin for 3 days. Thecultured cells were subjected to condi-tional culture for 3 days with or with-out treatment with 10 ng/ml of clus-terin protein purified from human

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serum (Kim et al., 2006). The culturemedium was substituted with freshmedium every day during the condi-tional culture.

Immunocytochemistry

Pancreatic tissue was fixed withBouin’s solution (saturated picricacid: formaldehyde, 3:1) with 0.1%acetic acid (Sigma-Aldrich, St. Louis,MO) for 24 hr at room temperatureand embedded in paraffin. Pancreaticsections 4–5 mm in thickness weredewaxed in xylene, and endogenousperoxidase activity was blocked using0.5% hydrogen peroxide in absolutemethanol for 30 min. Sections werethen washed in phosphate-bufferedsaline (PBS) and blocked with normalgoat serum or normal horse serum for30 min at room temperature. The tis-sue sections were incubated with a1:200 dilution of goat anti-clusterinantibody (Santa Cruz Biotechnology,Santa Cruz, CA), a 1:500 dilution ofrabbit anti-insulin (Santa Cruz Bio-technology), a 1:200 dilution of goatanti-cytokeratin 20 (Santa Cruz Bio-technology), a 1:100 dilution of rabbitanti-amylase (Sigma-Aldrich), and a1:150 dilution of mouse anti-PCNA(Millipore, Billerica, MA). Slidesstained for clusterin and CK-20 wereincubated with 10 mM citrate bufferfor 10 min at 90�C for antigen re-trieval before primary antibody appli-cation. Slides were incubated withprimary antibodies overnight at 4�Cbefore being washed in PBS and incu-bated with the secondary antibodies(biotinylated anti-mouse, anti-goat, oranti-rabbit IgG, and the avidin-biotin-peroxidase complex; Vector, Burlin-game, CA) for 1 hr at room tempera-ture. The reaction was developedusing diaminobenzidine tetrahydro-chloride (Sigma-Aldrich).

Cultured pancreatic duct cells werefixed with Bouin’s solution containing0.1% acetic acid for 5 min at roomtemperature. Endogenous peroxidaseactivity was blocked using absolutemethanol containing 0.5% hydrogenperoxide for 5 min at �20�C. For opti-mal immunolabelling, pancreatic ductcells were incubated with 0.5% tritonX-100 in PBS for 5 min and blockedwith normal goat serum for 30 min atroom temperature. Cultured cellswere incubated with a 1:500 dilution

of rabbit anti-insulin (1:500, SantaCruz Biotechnology), anti-PDX-1(1:500, Chemicon, Temecula, CA), oranti-MafA (Bethyl Laboratories,Montgomery, TX) overnight at �C. Forfluorescence staining, secondary anti-body was a 1:200 dilution of anti-rab-bit FITC (Vector, Burlingame, CA) for1 hr at room temperature. Stainedcells were mounted using VECTA-SHIELD mounting medium (Vector).Double immune-labeling was per-formed using pancreatic tissues andcultured cells. In order to avoid themismatching between the antibodiesfor double-lableing, two primary anti-bodies raised from different animalspecies were applied simultaneouslyand detected by corresponding sec-ondary antibodies labeled with FITCor TRITC. Double labeling was ana-lyzed using a confocal microscope(Carl Zeiss, Jena, Germany).

Real Time Quantitative

PCR Analysis

Real time quantitative PCR was car-ried out in an iCycler iQ multicolordetection system (Bio-Rad Laborato-ries) using the pancreatic tissue sam-ples of PPx-WT and PPx-CLU�/� at 6days after operation as well as fromthe un-operated WT controls. Reac-tions were performed in a 20-ml reac-tion volume using 2�iQ SYBR GreenSupermix (Bio-Rad Laboratories).The following primers were used;PTF1a-FWD 50-catagagaacgaaccaccctttgag-30; PTF1a-REV 50-gcacggagtttcctggacagagttc-30, Hes1-FWD 50-tctacaccagcaacagtg-30; Hes1-REV 50-tcaaacatctttggcatcac-30, Ngn3-FWD 50-tggcactcagcaaacagcga-30; Ngn3-REW 50-acccagagccagacaggtct-30, Isl1-FWD 50-agatatgggagacatgggcgat-30; Isl1-REW50-acacagcggaaacactcgatg-30, Pdx-1-FWD 50-ctcgctgggaacgctggaaca-30; Pdx-1-REW 50-gctttggtggatttcatccacgg-30,Nkx2.2-FWD 50-cacgcaggtcaagatctg-30;Nkx2.2-REW 50-tgcccgcctggaaggtggcg-30, Pax-4-FWD 50-tggctttctgtccttctgtgagg-30; Pax-4-REW 50-tccaagactcctgtgcggtagtag-30. Amplification was performedby initial polymerase activation for 10min at 95�C and 40 cycles of 95�C for30 s, 55�C for 30 s, and 72�C for 30 s.Quantitative values were obtained asthreshold PCR cycle number (Ct) whenthe increase in fluorescent signal ofPCR product showed exponential

amplification. The target gene mRNAlevel was normalized to that of18sRNA in the same sample. In brief,the relative expression level of the tar-get gene compared with that of18sRNA was calculated as 2�DCt,where DCt ¼ Ct target gene – Ct(18sRNA). The ratio of relative expres-sion of the target gene was calculatedas 2-(DDCt). Each sample was measuredin triplicate (Livak et al., 2001).

Intraperitoneal Glucose

Tolerance Test (IPGTT)

To examine the normal blood glucosevariations, IPGTT was performed inwild type and clusterin deficiencymice. Mice were intraperitoneallyinjected with 1 mg glucose/g bodyweight after fasting overnight (14–16hr). Blood samples were obtainedfrom the tail vein at 0, 5, 10, 15, 30,60, and 120 min and analyzed for glu-cose level using Mire 3.3G Glucome-ter (Infopia Co., Ltd, Gyeonggi-do,Korea).

Measurement of Insulin

Insulin levels in the pancreas and inthe serum were determined using amouse insulin ELISA kit (MercodiaAB, Uppsala, Sweden). Each samplewas diluted 1:10 and incubated onmouse monoclonal anti-insulin coatedplate. After incubation and washing,bound conjugates were detected byreaction with 3,30, 5,50-tetramethyl-benzidine. Color changes were ana-lyzed by a spectrophotometric micro-plate reader. Purified and knownconcentration calibrators were incu-bated parallel with unknown samplesfor standard curves.

Morphometry

To measure the area of regenerationor acinar-rich and duct-rich areas inpancreatic tissue, two paraffin sec-tions showing different tissue profilesin hematoxylin and eosin (H&E)staining were selected from each ani-mal sample. The sections were takenat intervals of approximately 200 par-affin sections. The tissue was photo-graphed with lower magnification(�10) to integrate a whole tissueimage using Photoshop program(Adobe Photoshop CS2. Microsoft Co.,

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San Jose, CA) (Supp. Fig. S2). Regen-erating pancreas was determined bymicroscopic observation and indicatedon the images. Since the margin ofthe regenerating lobules was notclearly demarcated due to the abun-dant fibrous tissue, we lined the out-ermost acini or ducts to figure outeach regenerating lobule in regener-ating areas as shown in Figure 1Gand H. The dimensions of each regen-erating area and total pancreatic areawere assessed using scion image pro-gram (Scion Corporation, Frederick,MA) (Fig. 1E). The data from 3 differ-ent animals in each groups were usedfor statistical analysis. For standardcalculation, each area size (mm2) wasconverted into percent (%) value. Esti-mation of duct-rich and acinar-richareas was similarly performed byregenerating area measurement.Briefly, the two areas were determinedby observation of double-stained sec-tion with CK-20 and amylase, or twoconsecutively stained sections, respec-tively. The areas were determined bythe structures demonstrating a pre-dominant immunoreactivity betweenCK-20 and amylase (Supp. Fig. S3).The areas were indicated on an inte-grated image of total pancreatic tissuefor assessment as described previously.

The number of insulin- or PCNA-positive cells in regenerating lobuleswas counted and presented as thenumber of immunoreactive cells/mm2.To determine regenerating activity ofthe pancreatic islet, total insulin-posi-tive beta-cells and newly developingislets with less than 6 beta-cells werecounted in regenerating lobules. Fordetermination of in vitro beta-cell dif-ferentiation by immunocytochemistry,we counted the insulin-positive cells ofthe explants in the immunocytochemi-cally-stained cover-slip (22 � 22 mm),which displayed 40–50 explants. Dataon differentiation are presented as thenumbers of insulin-positive cells in1,000 epithelial cells of the explants.

Statistical Analyses

All values are given as the mean 6S.E.M. Differences between twogroups were assessed using anunpaired two-tailed t-test. Data frommore than two groups were assessedby analysis of variance (ANOVA) fol-

lowed by a post-hoc least significantdifference test. Repeated ANOVA wasused for the studies of clusterinknockout and the stage of islet devel-opment (i.e., Fig. 3F and G). Statisti-cal analyses were performed usingSigmaPlot 10 (Systat Software, PointRichmond, CA).

ACKNOWLEDGMENTSThis work was supported by the KoreaScience and Engineering Foundationby the Ministry of Science and Tech-nology (M10642140004-06N4214-0040)to In-SunPark.

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