original article - diabetes€¦ · bcm measurements. parafﬁn sections (5 m) throughout the...

Original Article

Regulation of Pancreatic �-Cell Regeneration in theNormoglycemic 60% Partial-Pancreatectomy MouseMina Peshavaria, Brooke L. Larmie, James Lausier, Basanthi Satish, Aida Habibovic, Violet Roskens,

Kyla LaRock, Brian Everill, Jack L. Leahy, and Thomas L. Jetton

�-Cell mass is determined by a dynamic balance of prolif-eration, neogenesis, and apoptosis. The precise mecha-nisms underlying compensatory �-cell mass (BCM)homeostasis are not fully understood. To evaluate theprocesses that maintain normoglycemia and regulate BCMduring pancreatic regeneration, C57BL/6 mice were ana-lyzed for 15 days following 60% partial pancreatectomy(Px). BCM increased in Px mice from 2 days onwards andwas �68% of the shams by 15 days, partly due to enhanced�-cell proliferation. A transient �2.8-fold increase in theprevalence of �-cell clusters/small islets at 2 days post-Pxcontributed substantially to BCM augmentation, followedby an increase in the number of larger islets at 15 days. Toevaluate the signaling mechanisms that may regulate thiscompensatory growth, we examined key intermediates ofthe insulin signaling pathway. We found insulin receptorsubstrate (IRS)2 and enhanced-activated Akt immunoreac-tivity in islets and ducts that correlated with increasedpancreatic duodenal homeobox (PDX)1 expression. In con-trast, forkhead box O1 expression was decreased in isletsbut increased in ducts, suggesting distinct PDX1 regulatorymechanisms in these tissues. Px animals acutely adminis-tered insulin exhibited further enhancement in insulinsignaling activity. These data suggest that the IRS2-Aktpathway mediates compensatory �-cell growth by activat-ing �-cell proliferation with an increase in the number of�-cell clusters/small islets. Diabetes 55:3289–3298, 2006

The pancreatic �-cell has a substantial capacity tofunctionally compensate in response to physio-logical and pathophysiological changes in tissueinsulin requirements. A fundamental aspect of

this response is the dynamic regulation of �-cell mass(BCM). The steady-state BCM is influenced by a complexbalance of processes, which includes recruitment of newcells by hyperplasia of existing �-cells and neogenesis,apoptosis, and hypertrophy (1). Although the relativeimportance of these factors is unknown, analyses of

compensatory mechanisms are often complicated by hy-perglycemia, which can cause independent effects on�-cell gene expression, signaling, and BCM (2).

Recent studies in mice have concluded that �-cellsoriginate almost exclusively by proliferation from preex-isting �-cells (3–5). It has been suggested that islet numbermay be established early in life since �-cell turnover in ahealthy adult is quite low (6). In addition, using aninducible lineage-tracing technique to mark insulin promo-ter–transcribing cells, Dor et al. (3) observed that �-cellsappeared to derive solely from preexisting �-cells and thatthe number of islets after a 70% partial pancreatectomy(Px) remained static, with negligible contribution fromneogenesis. However, this issue remains controversial, asthe cellular origin(s) of new �-cells in the adult animal arenot well elucidated and depend on the animal model understudy. The importance of the insulin/IGF-1 pathway as amediator of BCM homeostasis has recently been under-scored in several studies using genetically altered miceeither deficient or overexpressing key elements of thispathway. It now appears that elements of the insulinsignaling pathway via IRS2, Pdk1, protein kinase B/Akt,and forkhead box O (FoxO)1 may regulate the expressionof the key transcription factor pancreatic duodenal ho-meobox (PDX)1 and, thus, mediate �-cell proliferation andfunction, size, and survival (7–16).

In this study, we have characterized �-cell regenerationfollowing a 60% Px in C56Bl/6 mice that also maintainnormoglycemia, probably due, in part, to an acute buttransient surge in �-cell growth and proliferation. Our datashow that �-cell growth and proliferation in response toPx is associated with enhanced IRS2/Akt/FoxO1 signalingin islet �-cells and a subset of epithelial cells in the ducts.

RESEARCH DESIGN AND METHODS

Sixty percent Px surgery. Sixty percent Px was performed on 8-week-oldmale C57BL/6 mice (n � 20–25 per group) (Taconic), as described previouslyfor rats (17) and mice (18). Mice were anesthetized with an intraperitonealinjection containing a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg).The surgery involved excision of the portion of the pancreas bordered by thespleen and stomach, extending to the small flap of pancreas attached to thepylorus, by gentle abrasion using cotton applicators. Control mice underwenta sham surgery involving laparotomy and gentle rubbing of the tissue. For allthese studies, the guidelines set forth by the University of Vermont Institu-tional Animal Care and Use Committee were strictly followed.Plasma glucose and insulin measurements. Fed whole-blood glucoselevels were measured with a glucose meter (Freestyle) and the plasmasubsequently analyzed for insulin utilizing a sensitive insulin radioimmunoas-say kit (Linco Research).Common pancreatic duct and islet isolation. Islets were isolated bycollagenase digestion and separated by histopaque density gradient centrifu-gation as described (19). Rat common pancreatic duct (CPD) was isolated insitu using a modified islet isolation procedure (20). Since both mouse and rat

From the Division of Endocrinology, Diabetes and Metabolism, University ofVermont, Burlington, Vermont.

Address correspondence and reprint requests to Mina Peshavaria, Univer-sity of Vermont College of Medicine, Department of Medicine, Given C331,Burlington, VT 05405. E-mail: [email protected].

Received for publication 4 January 2006 and accepted in revised form 30August 2006.

BCM, �-cell mass; CPD, common pancreatic duct; Foxa, Forkhead box A;FoxO, forkhead box O; IRS, insulin receptor substrate; pAkt, phospho-activated Akt; PDX, pancreatic duodenal homeobox; Px, partial pancreatec-tomy.

DOI: 10.2337/db06-0017© 2006 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

DIABETES, VOL. 55, DECEMBER 2006 3289

regenerating ducts show a similar expression pattern of insulin signalingintermediates, rat duct protein was used for immunoblotting to circumventlow abundance of protein from mouse ducts (17).Tissue processing for microscopy. Tissue processing for paraffin embed-ding was performed as described (21). For the detection of labile signalingintermediates, pancreata were rapidly dissected and then paraformaldehydefixed for 1.5 h, washed, equilibrated in 30% sucrose/PBS, and embedded inoptimal cutting temperature medium (Miles Scientific).BCM measurements. Paraffin sections (5 �m) throughout the entire pan-creas were mounted as ribbons on microscope slides to facilitate sectioncounting. Two or three slides (200-�m apart) from the broadest pancreaticsections were analyzed for BCM measurement (n � 5–10 for each group andtime point). These sections were found to be representative of the wholepancreas. Sections were immunostained for insulin, as described (21).

�-Cell fractional area was determined by digitally scanning entire sectionsusing a microslide scanner (Nikon Super CoolScan 9000). Image files wereprocessed in Adobe Photoshop and analyzed using NIH Image J (version133�), tabulated in pixel values converted to squared micrometers, andentered into Microsoft Excel for statistical analyses. Since the scannerafforded a maximal resolution of �40 �m2/pixel, we counted only insulin� cellclusters of �10 pixels (�400 �m2) or approximately three to four �-cells toexclude background “noise” in our measurements. BCM was estimated foreach animal by determining the fractional �-cell surface area per animalmultiplied by the pancreatic weight.Estimation of new �-cell formation based on cluster quantification. Therelative surface area and number of �-cell clusters and islets (�400 �m2) weretallied for each animal using the same sections used for the BCM measure-ments. �-Cell cluster size was initially categorized into one of six classes:400–599, 600–3,999, 4,000–7,999, 8,000–11,999 12,000–15,999, and �16,000�m2. �-Cell clusters (400–599 �m2) and small islets (600–3,999 �m2), regard-less of their location, were considered to be newly formed, possibly neogenic.For measuring �-cell clusters �400 �m2, we microscopically imaged 10 � 0.3mm2 fields for each animal and counted clusters of one to four cells.�-Cell, duct, and acinar cell proliferation. At least two slides per adultpancreas were stained for the cell proliferation marker, Ki-67, and insulin aspreviously detailed (21). The number of Ki-67� nuclei per 1,000–1,500 islet�-cells, per 300 CPD cells, and 10 � 0.3 mm2 fields of acinar tissue werecounted for each animal.Multiple-labeling immunofluorescence. Frozen sections of dissected CPDsand adjoining remnant pancreas were prepared for staining as described (21).The following primary antibodies were used: cyclin D2 (1:25 dilution),phospho-S473Akt (1:100), phosphotyrosine (1:500), phospho-FoxO1S256 (1:250), and phospho-S473Akt (1:100), all from Cell Signaling, and IRS2 (1:100),FoxO1 (1:250; Upstate), insulin (1:500; Linco), PDX1 (1:1,000; gift from Dr.Chris Wright, Vanderbilt University), Forkhead box A (Foxa)2 (1:30; SantaCruz), Ki-67 (1:500; BD Biosciences), and �-catenin (1:500; Zymed). Allsecondary antibodies contained “multiple labeling” grade anti-IgG conjugatedto CY2 (1:300), CY3 (1:2,000), or CY5 (1:500) (Jackson ImmunoResearch).Images were acquired using conventional epifluorescence or with a ZeissLSM510 confocal microscope (UVM Cell Imaging Facility). Images wereacquired with the LSM software and merged and formatted on a Macintosh G5with Adobe Photoshop.Immunoblotting and immunoprecipitation. Total-cell lysates from isolatedislets and CPDs were sonicated in lysis buffer consisting of 50 mmol/l HEPES(pH 7.5), 1% (vol/vol) Nonidet P-40, 2 mmol/l activated sodium orthovanadate,100 mmol/l sodium fluoride, 10 mmol/l sodium pyrophosphate, 1 mmol/lphenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche). Pro-tein content in the cell lysates was determined using the bicinchoninic acidprotein assay kit (Pierce). Cell lysates were separated by SDS-PAGE andtransferred to polyvinylidine flouride membranes. After blocking, membraneswere incubated in appropriate primary antibody followed by horseradishperoxidase–conjugated secondary antibodies. Positive signals were visualizedwith chemiluminescence (Pierce). For immunoprecipitation experiments,lysates were incubated with anti-Akt1/2 overnight and immunoblots probedwith phospho-activated Akt (pAkt) antibody.Semiquantitative radioactive duplex PCR. Total RNA was extracted fromisolated islets and collagenase digested CPDs using Trizol reagent (Invitro-gen). cDNA was synthesized using the first-strand cDNA synthesis kit (RocheApplied Science). PCR analyses were conducted using a previously describedprotocol (20). Typically, 20–30 cycles were run to maintain the amplificationwithin the linear range. (Primer sequences for FoxO1 and Foxa2 are availablefrom the authors upon request.) An 18S rRNA primer pair (Ambion, Austin,TX) was used as an internal control for PCR analysis. PCR products wereseparated on a 6% polyacrylamide gel in Tris-borate EDTA buffer. The gel wasexposed to a PhosphoImager screen and band intensities quantitated with aBioRad Molecular PhosphoImager (UVM DNA Analysis Core Facility).

RESULTS

General metabolic characteristics of Px mice. Dailyblood samples from 60% Px and sham-operated C57BL/6mice were analyzed for nonfasting glucose and insulinconcentrations over a period of 15 days postsurgery andwere not significantly different at any time point betweenthe groups (Fig. 1A and B). Both groups also maintainedidentical weights throughout the period (data not shown).BCM and islet proliferation in 60% Px mice. Wemeasured BCM at 2, 6, and 15 days post-Px. Although notsignificant, sham-operated mice exhibited the expectedincreases in BCM for 8- to 10-week-old mice commensu-rate with age-related weight increases during the 2-weekstudy period compared with unoperated age-matchedmice (Fig. 1C). In the Px mice, BCM increased progres-sively from �46, 59, and 68% relative to shams at 2, 6, and15 days post-Px, respectively. We next examined the basisfor the increase in BCM by measuring �-cell mitoticfrequencies. Although there was no significant increase at2 days post-Px, �-cell proliferation frequency increased2.5-fold over shams at 6 days post-Px and then returned tothat of the shams by 15 days (Fig. 1D). Six days postsur-gery, sham-operated mice also exhibited a small butsignificant increase in �-cell proliferation compared withthe 2-day sham group; however, both sham and Px groupsshowed depressed �-cell proliferative activity at 2 dayscompared with untouched mice, likely due to suppressedfeeding activity following laparotomy (Fig. 1D). Cyclin D2,a member of the D-type cyclin family, plays a crucial rolein �-cell proliferation (4,5). We detected a marked increasein cyclin D2 protein by immunoblotting and immunoreac-tivity, with an increase in the number of �-cell nucleiexpressing the protein in 6 days post-Px mice comparedwith sham mice (Fig. 1E). No differences were observed incyclin D2 expression in the 15-day groups (data not shown).BCM enhancement is associated with a transient

increase in small �-cell clusters. Although controver-sial, �-cell neogenesis has been proposed to play animportant role in BCM compensation (10,17,20–23). Wetherefore quantified the prevalence of �-cell clusters andislets and ranked them according to size to determine theircontribution to BCM increase in C57BL/6 mice after Px.We observed an �2.8-fold higher incidence of �-cell clus-ters/small islets (1,000- to 4,000-�m2 range) in 2-day Pxmice compared with sham controls (Fig. 1F). In contrast,we detected no increase in the number of very small �-cellclusters (one to four cells in cross-section) between the 2and 6 day postsurgery groups at this time point (data notshown). Proliferation was also rare in both �-cell clustersizes with no differences detected between groups. Wenext quantified the prevalence of islet sizes ranging�4,000–8,000, 8,000–11,960, 12,000–16,000, and �16,000�m2 (Table 1). We detected an increase of about twofoldin the larger 8,000–11,960 and 12,000–16,000 �m2 islet sizegroups at 15 days postsurgery (islet diameter range �90–125 �m) (Table 1). Collectively, these results seem consis-tent with an early postmitotic differentiation of �-cellprecursors, which initially appear as small �-cell clustersand then subsequently proliferate to larger islets.Enhanced BCM in 60% Px mice correlates with in-creased Akt signaling and PDX1 expression in pan-creatic �-cells. To determine if the IRS2/Akt/PDX1signaling pathway is activated during the BCM increase,we analyzed the expression of these proteins at 6 dayspostsurgery when �-cell proliferation is maximal. By im-

�-CELL MASS COMPENSATION IN THE Px MOUSE

3290 DIABETES, VOL. 55, DECEMBER 2006

munostaining, no differences were observed in �-cell IRS2levels between groups (data not shown). Immunostainingand immunoblot analyses revealed low to moderate levelsof pAkt in islet �-cells of control mice (Fig. 2A and C).

However, surface-oriented pAkt immunoreactivity wasenhanced in islets of 6-day Px mice (Fig. 2B and C).Furthermore, pAkt expression correlated with increasedlevels of PDX1 expression in �-cells (Fig. 2B and C).

FIG. 1. Metabolic and �-cell growth characteristics in 60% Px mice. Fed blood glucose (A) and insulin concentrations (B) were comparablebetween sham and Px mice through 15 days (d) postsurgery (n > 10). Values are means � SE. C: BCM increased progressively during the 2 weekspostsurgery in Px mice compared with sham controls. Untouched 2- and 15-day mice are shown for comparison. The black line superimposed onthe sham bars represents the theoretical BCM postsurgery. *P < 0.01; **P < 0.001; n > 5. D: Compared with shams, �-cell proliferation increased2.54-fold in the 6 days post-Px mice but was unchanged at 2 and 15 days. *P < 0.03; **P < 0.001; n > 5. E: A wide-field immunofluorescence imageand immunoblot showing increased cyclin D2 signal in 6-day Px islet compared with sham controls. F: A 2.8-fold increase in �-cell clusters andsmall islets was observed in 2-day Px mice compared with sham animals. *P < 0.05.

M. PESHAVARIA AND ASSOCIATES


Decreased FoxO1 but enhanced Foxa2 expression inregenerating �-cells. The forkhead transcription factorFoxO1 plays key roles in regulating apoptosis, prolifera-tion, and glucose metabolism. The function of FoxO1 isinhibited by Akt-mediated phosphorylation and its subse-quent nuclear exclusion (24). Recent studies suggest that

FoxO1 regulates BCM by inhibiting Pdx1 gene transcrip-tion through Foxa2 binding (12). We therefore examinedFoxO1 and Foxa2 expression in mice at 2 and 6 dayspost-Px. Compared with sham mice, we observed a mod-erate decrease in total cytoplasmic FoxO1 by immuno-staining and immunoblotting and a marked decrease in

FIG. 2. Enhanced pAkt, PDX1, and Foxa2, but decreased FoxO1, expression in Px mouse islets. A and B: Surface pAkt and nuclear PDX1immunoreactivity were increased in Px islets compared with low levels detected in 6-day sham islets. C: Islets from representative 6-day animalswere immunoprecipitated with Akt1/2 and immunoblotted with pAkt antibody (upper panel) and probed with PDX1 and actin antibodies (lower

panel). D and E: FoxO1 levels were decreased in 6-day Px �-cells compared with sham islets. Peripheral PDX1�� cells, and some PP� cells,stained intensely for FoxO1. F: Representative sham and Px islets from 2 and 6 days were probed with phospho-activated FoxO1 antibodyfollowed by total FoxO1 antibody. G and H: A wide-field immunofluorescence image of representative islets showing modest increases in Foxa2in 6-day Px islet compared with shams.

TABLE 1Distribution of medium to large islets in 60% sham and Px mice

Prevalence/cm2 of medium to large size class islets4,000–8,000 8,000–11,960 12,000–16,000 �16,000

2-day sham 0.063 0.012 0.025 0.007 0.026 0.008 0.015 0.0072-day Px 0.085 0.027 0.031 0.013 0.031 0.007 0.010 0.0056-day sham 0.069 0.010 0.030 0.005 0.030 0.005 0.016 0.0046-day Px 0.085 0.019 0.035 0.007 0.039 0.011 0.026 0.00915-day sham 0.096 0.012 0.029 0.006 0.028 0.006 0.016 0.00415-day Px 0.113 0.019 0.059 0.010* 0.055 0.011* 0.037 0.013

Data are means SE (n � 9–10 animals for each time point analysis). *P � 0.05 compared with sham controls.



phospho-FoxO1 by immunoblotting in 6-day Px mice (Fig.2D–F). Hence, it appears that both the total and phosphor-ylated (presumably cytoplasmic) pools of �-cell FoxO1 arereduced in 6-day Px mice. No changes were detected instrongly FoxO1� peripheral cells that were often PDX1�/somatostatin� (Fig. 2D and E).

We next analyzed the expression of Foxa2 in 6-day Pxmice by immunostaining. We detected moderate increasesin �-cell nuclear Foxa2 immunoreactivity compared withsham-operated mice (Fig. 2G and H).Heightened proliferation in the CPD epithelium inthe regenerating pancreas. Our previous studies in therat 60% Px model have shown that enhanced CPD prolif-eration was highest in the lateral evaginations as opposedto main epithelial cell lining (17). Accordingly, by immu-nostaining with Ki-67, we observed increased frequency ofstaining in the evaginations of 2-day Px mice (Fig. 3B andD) compared with shams (Fig. 3A and C). We quantifiedCPD proliferation, distinguishing between the main epithe-lial lining versus the evaginations. Compared with controlmice, we detected a 10-fold increase in the mitotic fre-quency of the evaginations at 2 days post-Px (P � 0.001),which was sustained at 6 days post-Px (Fig. 3E). Althoughinsulin� cells in Px mice were associated with evagina-tions, their low frequency precluded a quantitative analysis.Augmented insulin signaling in ducts of Px mice. New�-cell development, or neogenesis, is widely considered tooriginate from the pancreatic exocrine ducts (17,20,25–27). Hence, we next sought to examine the activity of theIRS2/Akt/FoxO1 pathway in the common pancreatic ductepithelium, since we detected transient increases in thenumber of small islets/�-cell clusters early in the regener-ation period along with hyperproliferation of cells liningthe duct evaginations. Our previous studies in the 60% Pxrat model had demonstrated enhanced IRS2 and pAktimmunoreactivity in the CPD during pancreatic regenera-tion that correlated with �-cell neogenesis (17). Similarly,we also observed strong IRS2 staining in the CPD evagi-nations of 2- and 6-day Px mice that normalized by 15 dayspostsurgery (Fig. 4A and B). pAkt immunoreactivity wasalso increased at 2 and 6 days post-Px compared withsham ducts (Figs. 3A and B and 4C–E). Surface-orientedpAkt immunoreactivity correlated with increased nuclearPDX1 expression in Px mice, especially in the duct evagi-nations (Figs. 3D and 4D). Correspondingly, by immuno-blotting, PDX1 protein levels were increased �1.5-fold in2-day Px mice; however, pAkt levels were markedly en-hanced at both 2 and 6 days post-Px in CPD extracts (Fig.4E).

Since recent reports have demonstrated FoxO1 expres-sion in pancreatic ducts and because it antagonizes Foxa2function in islets (12), we first examined FoxO1 and Foxa2mRNA by RT-PCR in ducts and islets of Px rats. Anincrease of �1.5-fold in FoxO1 and Foxa2 mRNA levelsover sham controls was observed in isolated CPDs from2-day Px rats, while there was a marginal but significantdecrease in islets (Fig. 5A and B). By immunostaining andimmunoblotting, intense cytoplasmic FoxO1 (Fig. 5C, D,and G) and nuclear Foxa2 immunoreactivity (Fig. 5E andF) were observed in the regenerating duct epithelium,especially in the evaginations. The contrasting profiles ofFoxO1 mRNA and protein levels in islets and ducts suggestdifferent regulatory mechanisms in these tissues.In vivo insulin administration to Px mice augments�-cell Akt signaling. To compare the status of the insulinsignaling intermediates between a chronic (i.e., Px sur-

gery) and short-term stimulus, insulin (750 mU/kg i.p.) orsaline was administered to 6-day sham and Px mice andthen analyzed at intervals for IRS2/pAkt/FoxO1 and PDX1immunoreactivity in islets and ducts. At 10 min postinjec-tion, although increased surface-oriented pAkt was ob-served in �-cells of saline-treated Px mice compared withshams (Fig. 6A and B), there was a striking enhancementin cytoplasmic pAkt in insulin-treated Px �-cells comparedwith sham islets (Fig. 6C and D). Unexpectedly, we alsoobserved enhanced IRS2 immunoreactivity in the islets ofinsulin-treated Px mice compared with shams (Fig. 6E andF). FoxO1 immunoreactivity was drastically reduced ininsulin-treated Px mice compared with insulin-treated

FIG. 3. Proliferation in the CPD evagination is enhanced in 2-day 60%Px mice. A: Decreased number of Ki-67� cells and low pAkt wereobserved in the sham duct lining and evaginations (arrow). B: Astriking increase in Ki-67�/pAkt� cells, concentrated in the evagina-tions (arrows), was observed in the Px ducts. C: High magnificationfield of an evagination (arrow) in shams with little mitotic activity, asopposed to several PDX1�/Ki-67� cells (D) in evaginations of Px mice(arrow). A–D: *Duct lumen. E: In contrast to common duct epitheliallining, proliferation frequency in the 2-day Px animals, compared withshams, is substantially increased in evaginations (*P < 0.001). Amoderate increase was observed in 6-day Px evaginations, whichnormalized by 15 days.



sham mice (Fig. 6G and H). In contrast, increased FoxO1immunoreactivity was observed in the CPD evaginationsof insulin-treated Px mice compared with a correspondingtreatment of control mice (Fig. 6I and J). These resultsshow that the observed pattern of tissue-specific signalingintermediate expression and activation noted post-Px wasaugmented by exogeneous insulin.

DISCUSSION

Although the metabolic characteristics of compensatorypartial regeneration of the pancreatic �-cells have beenwell characterized in the Px rodent model (17,28,29), adetailed study of the underlying mechanisms that areresponsible for the BCM expansion have not been previ-ously reported. Previous studies in C57BL/6 (18) and othermouse strains (30–32) following Px have highlighted theimpact of �-cell proliferation in BCM homeostasis. On theother hand, pancreatic duct ligation (33) and glucose-infusion rodent models (34) have emphasized BCM con-tributions by neogenesis originating from exocrine tissues.However, the confounding effects of hyperglycemia inthese models on BCM compensation, the signaling path-

ways, and �-cell gene expression could not be excluded(2). In contrast, the 60% Px rodent model offers a signifi-cant advantage in examining BCM compensation since avigorous, but transient, regeneration process ensues de-spite a 60% reduction in BCM (17,18).

In this study, we have investigated the mechanisms thatmediate BCM expansion in 60% Px C57BL/6 mice. Therewas a net increase in BCM of �28% versus shams by 15days following Px due to an increase in the number of�-cell clusters/small islets, possibly from an early postmi-totic differentiation of as yet unidentified �-cell precur-sors, followed by enhanced �-cell proliferation inpreexisting islets. We have demonstrated that these con-secutive processes correlate strongly with enhanced insu-lin receptor pathway signaling, with Akt kinase serving asa central intermediate.The role of insulin signaling–mediated duct prolifer-ation and differentiation in BCM compensation. Pre-vious studies in both 60 and 90% Px rat models have shownthat proliferation preceded differentiation in the epithe-lium of the large ducts (17,28). We detected a massive10-fold increase in proliferation in the epithelial lining ofthe lateral evaginations of the CPD at 2 days post-Px.Concomitant with an increase in proliferation, we ob-served a transient activation of the insulin signaling path-way in the CPD, as evidenced by increased IRS2, pAkt, andFoxO1 immunoreactivity in the C57BL/6 mice post-Px inconcert with enhanced PDX1 expression. Thus, in 2-daypost-Px mice, PDX1, IRS2, activated Akt, FoxO1, andFoxa2 were all expressed in the CPD lining; however, theywere more robustly expressed in the epithelial cells of theduct evaginations versus the lining. More importantly,strong pAkt� cells correlated with intense nuclear PDX1expression. However, since very few insulin� cells weredetected within the ducts of the sham and Px mice, it isunclear whether these strong pAkt�/PDX1� cells progressto fully differentiated �-cells to contribute to the islet�-cell pool.

In parallel with a peak in duct proliferation and en-hanced Akt signaling at 2 days post-Px, there was ashort-lived 2.8-fold increase in the number of �-cell clus-ters and small islets. As the prevalence of these clusterswaned, �-cell proliferation peaked at 6 days post-Px,correlating with a 50% increase in BCM. By 15 dayspostsurgery, increased numbers of large islets were ob-served in Px mice, suggesting continued growth of the islet�-cell population following the transient �-cell hyperpro-liferation occurring 1 week prior. Although there arecurrently no specific markers for newly differentiatedversus newly replicated �-cells, these results suggest thatin this mouse model of Px, BCM expansion involves notonly the expected proliferation from preexisting �-cellsbut possibly from newly formed islets. The contribution of�-cell neogenesis from ducts or other pancreatic progeni-tor cells to both normal BCM homeostasis (1,35) and to anexperimental regeneration stimulus (17,25,34) has recentlybeen challenged by studies reporting that the primarymechanism of �-cell renewal is by proliferation of preex-isting �-cells (3). Although our results strongly suggestthat the early increase in the number of �-cell clusterslikely contributes to BCM compensatory increase, thesource of these �-cell clusters/small islets remains un-known, as we failed to detect proliferating �-cell clusters,and the frequency of insulin� cells in the duct epitheliumwas too low to be quantified. However, increased numbersof single/double �-cells have been observed in transgenic

FIG. 4. Increased insulin signaling activity and PDX1 expression in2-day Px mouse duct evaginations. Although low levels of IRS2 andphosphotyrosine (pY) (A) were detected in sham duct lining andevaginations (arrow), increased IRS2 levels (B), with frequently morepY� cells, were observed in the evaginations of Px mice (arrow). C:Occasionally, cells expressing high levels of nuclear PDX1 and pAkt(inset) were seen in sham ducts; however, evaginations of Px mice (D)exhibited a striking correlation between heightened levels pAkt andPDX1 staining (arrows). A–D: *Duct lumen. E: A representative immu-noblot of 2- and 6-day sham and Px ducts probed with PDX1, pAkt, andtotal Akt antibodies. �-Catenin was used as a loading control.



mice overexpressing constitutively active Akt1 (9). Thus,in the current study, BCM expansion due to Akt activationmay not only regulate hyperproliferation of existing �-cellsbut, possibly, �-cell differentiation from exocrine progen-itors as well.Role of proliferation during BCM homeostasis. Stud-ies of mice with altered gene expression suggest that theinsulin signaling pathway via Akt/FoxO1/PDX1 regulates�-cell proliferation (7,12,14–16). In turn, the �-cell prolif-erative response is associated with increased levels of cellcycle proteins, including cyclin D1, cyclin D2, p21, and

cdk4 activity mediated by Akt (4,5,36,37). Thus, whereasFoxO1 haplodeficiency restored Pdx1 expression and�-cell proliferation in IRS2/ and Pdk1/ mice (12,15),PDX1 haplodeficiency abrogated �-cell compensatory re-sponse in insulin-resistant Insr�//Irs1�/ mice and inmice lacking insulin receptor in liver through impaired�-cell–proliferation mice (14). These results suggest thatFoxO1 and PDX1 may mediate proliferative signalsthrough Akt.

A universal finding in normal rodent Px models is theseveral-fold increase in �-cell proliferation peaking 4–7

FIG. 5. Enhanced FoxO1 and Foxa2 in mouse Px duct evaginations. A: Semiquantitative RT-PCR analysis showing increased FoxO1 mRNA levelsin rat ducts but a corresponding modest decrease in islets. B: An identical pattern between ducts and islets was observed with Foxa2 mRNA. *P �0.05; n > 3. C: A representative field of a sham duct showing undetectable levels of FoxO1 staining in the evaginations (arrows). D: A comparablefield from a Px mouse displaying heightened FoxO1 staining in evaginations. E: Moderate levels of nuclear Foxa2 staining were observed in themain lining and evaginations (arrow) of sham mice. F: In the Px mice CPDs, a global increase was seen of Foxa2 staining in the lining but witha further enhancement in evaginations (arrows). C–F: *Lumen. G: Ducts from representative 2- and 6-day sham and Px mice were probed withpFoxO1 antibody. Actin served as a loading control.



days postsurgery. Individual islet growth, including thatfollowing a Px stimulus, would be expected to be aproduct of �-cell proliferation, hypertrophy, apoptosis,and clearance (1,17,38). This was suggested by a 2.5-foldenhancement in �-cell proliferation at 6 days post-Px,concomitant with increased cyclin D2 expression. Thus,enhanced Akt signaling in �-cells from 6 days post-Px micelikely mediates this heightened replication.Insulin signaling mediates BCM homeostasis in Pxmice. Recent studies indicate that the IRS2/Akt/FoxO1signaling pathway plays a pivotal role in compensatoryBCM augmentation in insulin-resistance states, with �-cellproliferation as a primary mechanism for maintenance ofthis BCM (7–16,39). Rescue of diabetes in Pdk1/ andIrs2/ mice by superimposing FoxO1 haploinsufficiency,and by over-expression of PDX1 in Irs2/ mice, essen-tially restored BCM and PDX1 expression, implying aregulatory link between the insulin signaling cascade andPDX1 (12,13,15). Although the impact of this pathway inresponse to �-cell regeneration is not yet fully resolved,we have previously reported in the 60% rat Px model thatincreased IRS2 and pAkt levels correlated with enhancedneogenesis from the large ducts (17). While IRS2 levels in�-cells were not significantly changed during �-cell regen-eration, possibly due to degradation (40), �-cell pAktimmunoreactivity was intense and surface oriented and, at6 days post-Px, correlated strongly with nuclear PDX1. Akthas been ascribed roles in regulating �-cell size, survival,proliferation (9–11,41,42), and neogenesis (9,17). Many ofthese functions are likely manifestations of Akt signalingthrough the transcription factor FoxO1 and, in turn, PDX1.In islets of Px mice, cytoplasmic FoxO1 expression wasdecreased profoundly in �-cells of 6-day Px mice andcorrelated with enhanced PDX1 levels. Although not sub-stantiated, these observations suggest that phosphorylatedFoxO1 may be rapidly degraded following nuclear exclu-sion and PDX1 activation (12,43). In contrast to islets,increased cytoplasmic expression of FoxO1 correlatedwith increased nuclear PDX1 expression in the CPDevaginations. This observation was puzzling, since weanticipated decreased cytoplasmic FoxO1 immunoreactiv-ity due to intense nuclear PDX1 and Foxa2 immunoreac-tivity in the CPD evaginations. Although unclear, theregulation of FoxO1 activity appears tissue specific andwarrants further investigation.

We have also detected moderate increases in Foxa2mRNA and protein levels in Px mice consistent with itsrole as an activator of PDX1 expression (44). As PDX1 hasrecently been ascribed roles in �-cell survival and prolif-eration (14,45), these results support our premise thatenhanced BCM in Px mice involves �-cell signalingthrough Akt, FoxO1, and PDX1, regulating �-cell growth.Our findings that a transient stimulation of the insulinsignaling pathway by insulin itself further enhanced theexpression pattern of signaling elements during �-cellregeneration underscores the impact of this pathway inBCM expansion.

In conclusion, the 60% Px model serves as a usefulparadigm for examining the mechanisms and signalingpathways for �-cell regeneration following pancreatic in-jury. The current study in the C57BL/6 mouse straindemonstrates that �-cell regeneration entails coordinatedprocesses that involve an early increase in the prevalenceof �-cell clusters with no demonstrable proliferation ofthese insulin� cells. Subsequently, �-cell hyperprolifera-tion occurs as a second-phase response to the �-cell

FIG. 6. Augmented insulin signaling activity upon insulin administrationin 6 days post-Px mice. Compared with saline-injected sham islets (A),saline-injected Px islets (B) exhibited an increase in nuclear PDX1 andsurface-oriented pAkt immunostaining. However, compared with insulin-treated sham islets (C), cytoplasmic pAkt immunostaining was enhancedseveral-fold in insulin-injected Px islets (D). Low level of cytoplasmicIRS2 staining was observed in insulin-injected sham islets (E); however,cytoplasmic IRS2 increased dramatically in insulin-injected Px islets (F).Moderate FoxO1 staining in �-cells was observed in insulin-treated shams(G), which was undetectable after insulin administration (H). I: Lowlevels of diffuse cytoplasmic FoxO1 immunostaining were detectedthroughout the duct lining and evaginations (arrow) in insulin-treatedsham mice. J: Increased FoxO1 was seen in evaginations (arrows) of Pxmice, although diffuse levels were maintained in the duct lining.



regeneration stimulus. The IRS2/Akt/FoxO1/PDX1 path-way appears to mediate this �-cell growth response. Wespeculate that an early response involving increased insu-lin signaling activity and proliferation in pancreatic ductspost-Px may be related to neogenesis.

ACKNOWLEDGMENTS

This work was supported by a CDA from the JuvenileDiabetes Research Foundation and a Research Awardfrom the American Diabetes Association (to M.P.) and bythe National Institutes of Health (grant DK-068329 to T.L.J.and grants DK-56818 and DK-66635 to J.L.L.).

We acknowledge Dr. Dhananjay Gupta for the criticalreading of the manuscript. We also are indebted to Dr.Afshin Salsali who helped in the initiation of this project.

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