β-Cell mass is determined by a dynamic balance of proliferation, neogenesis, and apoptosis. The precise mechanisms underlying compensatory β-cell mass (BCM) homeostasis are not fully understood. To evaluate the processes that maintain normoglycemia and regulate BCM during pancreatic regeneration, C57BL/6 mice were analyzed for 15 days following 60% partial pancreatectomy (Px). BCM increased in Px mice from 2 days onwards and was ∼68% of the shams by 15 days, partly due to enhanced β-cell proliferation. A transient ∼2.8-fold increase in the prevalence of β-cell clusters/small islets at 2 days post-Px contributed substantially to BCM augmentation, followed by an increase in the number of larger islets at 15 days. To evaluate the signaling mechanisms that may regulate this compensatory growth, we examined key intermediates of the insulin signaling pathway. We found insulin receptor substrate (IRS)2 and enhanced-activated Akt immunoreactivity in islets and ducts that correlated with increased pancreatic duodenal homeobox (PDX)1 expression. In contrast, forkhead box O1 expression was decreased in islets but increased in ducts, suggesting distinct PDX1 regulatory mechanisms in these tissues. Px animals acutely administered insulin exhibited further enhancement in insulin signaling activity. These data suggest that the IRS2-Akt pathway mediates compensatory β-cell growth by activating β-cell proliferation with an increase in the number of β-cell clusters/small islets.

The pancreatic β-cell has a substantial capacity to functionally compensate in response to physiological and pathophysiological changes in tissue insulin requirements. A fundamental aspect of this response is the dynamic regulation of β-cell mass (BCM). The steady-state BCM is influenced by a complex balance of processes, which includes recruitment of new cells by hyperplasia of existing β-cells and neogenesis, apoptosis, and hypertrophy (1). Although the relative importance of these factors is unknown, analyses of compensatory mechanisms are often complicated by hyperglycemia, which can cause independent effects on β-cell gene expression, signaling, and BCM (2).

Recent studies in mice have concluded that β-cells originate almost exclusively by proliferation from preexisting β-cells (35). It has been suggested that islet number may be established early in life since β-cell turnover in a healthy adult is quite low (6). In addition, using an inducible lineage-tracing technique to mark insulin promoter–transcribing cells, Dor et al. (3) observed that β-cells appeared to derive solely from preexisting β-cells and that the number of islets after a 70% partial pancreatectomy (Px) remained static, with negligible contribution from neogenesis. However, this issue remains controversial, as the cellular origin(s) of new β-cells in the adult animal are not well elucidated and depend on the animal model under study. The importance of the insulin/IGF-1 pathway as a mediator of BCM homeostasis has recently been underscored in several studies using genetically altered mice either deficient or overexpressing key elements of this pathway. It now appears that elements of the insulin signaling pathway via IRS2, Pdk1, protein kinase B/Akt, and forkhead box O (FoxO)1 may regulate the expression of the key transcription factor pancreatic duodenal homeobox (PDX)1 and, thus, mediate β-cell proliferation and function, size, and survival (716).

In this study, we have characterized β-cell regeneration following a 60% Px in C56Bl/6 mice that also maintain normoglycemia, probably due, in part, to an acute but transient surge in β-cell growth and proliferation. Our data show that β-cell growth and proliferation in response to Px is associated with enhanced IRS2/Akt/FoxO1 signaling in islet β-cells and a subset of epithelial cells in the ducts.

Sixty percent Px surgery.

Sixty percent Px was performed on 8-week-old male C57BL/6 mice (n = 20–25 per group) (Taconic), as described previously for rats (17) and mice (18). Mice were anesthetized with an intraperitoneal injection 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 the spleen and stomach, extending to the small flap of pancreas attached to the pylorus, by gentle abrasion using cotton applicators. Control mice underwent a sham surgery involving laparotomy and gentle rubbing of the tissue. For all these studies, the guidelines set forth by the University of Vermont Institutional Animal Care and Use Committee were strictly followed.

Plasma glucose and insulin measurements.

Fed whole-blood glucose levels were measured with a glucose meter (Freestyle) and the plasma subsequently analyzed for insulin utilizing a sensitive insulin radioimmunoassay kit (Linco Research).

Common pancreatic duct and islet isolation.

Islets were isolated by collagenase digestion and separated by histopaque density gradient centrifugation as described (19). Rat common pancreatic duct (CPD) was isolated in situ using a modified islet isolation procedure (20). Since both mouse and rat regenerating ducts show a similar expression pattern of insulin signaling intermediates, rat duct protein was used for immunoblotting to circumvent low abundance of protein from mouse ducts (17).

Tissue processing for microscopy.

Tissue processing for paraffin embedding was performed as described (21). For the detection of labile signaling intermediates, pancreata were rapidly dissected and then paraformaldehyde fixed for 1.5 h, washed, equilibrated in 30% sucrose/PBS, and embedded in optimal cutting temperature medium (Miles Scientific).

BCM measurements.

Paraffin sections (5 μm) throughout the entire pancreas were mounted as ribbons on microscope slides to facilitate section counting. Two or three slides (200-μm apart) from the broadest pancreatic sections were analyzed for BCM measurement (n = 5–10 for each group and time point). These sections were found to be representative of the whole pancreas. Sections were immunostained for insulin, as described (21).

β-Cell fractional area was determined by digitally scanning entire sections using a microslide scanner (Nikon Super CoolScan 9000). Image files were processed in Adobe Photoshop and analyzed using NIH Image J (version 133μ), tabulated in pixel values converted to squared micrometers, and entered into Microsoft Excel for statistical analyses. Since the scanner afforded a maximal resolution of ∼40 μm2/pixel, we counted only insulin+ cell clusters of >10 pixels (∼400 μm2) or approximately three to four β-cells to exclude background “noise” in our measurements. BCM was estimated for each animal by determining the fractional β-cell surface area per animal multiplied by the pancreatic weight.

Estimation of new β-cell formation based on cluster quantification.

The relative surface area and number of β-cell clusters and islets (>400 μm2) were tallied for each animal using the same sections used for the BCM measurements. β-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 μm2. β-Cell clusters (400–599 μm2) and small islets (600–3,999 μm2), regardless of their location, were considered to be newly formed, possibly neogenic. For measuring β-cell clusters <400 μm2, we microscopically imaged 10 × 0.3 mm2 fields for each animal and counted clusters of one to four cells.

β-Cell, duct, and acinar cell proliferation.

At least two slides per adult pancreas were stained for the cell proliferation marker, Ki-67, and insulin as previously detailed (21). The number of Ki-67+ nuclei per 1,000–1,500 islet β-cells, per 300 CPD cells, and 10 × 0.3 mm2 fields of acinar tissue were counted for each animal.

Multiple-labeling immunofluorescence.

Frozen sections of dissected CPDs and 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; Santa Cruz), Ki-67 (1:500; BD Biosciences), and β-catenin (1:500; Zymed). All secondary antibodies contained “multiple labeling” grade anti-IgG conjugated to CY2 (1:300), CY3 (1:2,000), or CY5 (1:500) (Jackson ImmunoResearch). Images were acquired using conventional epifluorescence or with a Zeiss LSM510 confocal microscope (UVM Cell Imaging Facility). Images were acquired with the LSM software and merged and formatted on a Macintosh G5 with Adobe Photoshop.

Immunoblotting and immunoprecipitation.

Total-cell lysates from isolated islets 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/l phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche). Protein content in the cell lysates was determined using the bicinchoninic acid protein assay kit (Pierce). Cell lysates were separated by SDS-PAGE and transferred to polyvinylidine flouride membranes. After blocking, membranes were incubated in appropriate primary antibody followed by horseradish peroxidase–conjugated secondary antibodies. Positive signals were visualized with chemiluminescence (Pierce). For immunoprecipitation experiments, lysates were incubated with anti-Akt1/2 overnight and immunoblots probed with phospho-activated Akt (pAkt) antibody.

Semiquantitative radioactive duplex PCR.

Total RNA was extracted from isolated islets and collagenase digested CPDs using Trizol reagent (Invitrogen). cDNA was synthesized using the first-strand cDNA synthesis kit (Roche Applied Science). PCR analyses were conducted using a previously described protocol (20). Typically, 20–30 cycles were run to maintain the amplification within the linear range. (Primer sequences for FoxO1 and Foxa2 are available from the authors upon request.) An 18S rRNA primer pair (Ambion, Austin, TX) was used as an internal control for PCR analysis. PCR products were separated on a 6% polyacrylamide gel in Tris-borate EDTA buffer. The gel was exposed to a PhosphoImager screen and band intensities quantitated with a BioRad Molecular PhosphoImager (UVM DNA Analysis Core Facility).

General metabolic characteristics of Px mice.

Daily blood samples from 60% Px and sham-operated C57BL/6 mice were analyzed for nonfasting glucose and insulin concentrations over a period of 15 days postsurgery and were not significantly different at any time point between the groups (Fig. 1A and B). Both groups also maintained identical weights throughout the period (data not shown).

BCM and islet proliferation in 60% Px mice.

We measured BCM at 2, 6, and 15 days post-Px. Although not significant, sham-operated mice exhibited the expected increases in BCM for 8- to 10-week-old mice commensurate with age-related weight increases during the 2-week study period compared with unoperated age-matched mice (Fig. 1C). In the Px mice, BCM increased progressively from ∼46, 59, and 68% relative to shams at 2, 6, and 15 days post-Px, respectively. We next examined the basis for the increase in BCM by measuring β-cell mitotic frequencies. Although there was no significant increase at 2 days post-Px, β-cell proliferation frequency increased 2.5-fold over shams at 6 days post-Px and then returned to that of the shams by 15 days (Fig. 1D). Six days postsurgery, sham-operated mice also exhibited a small but significant increase in β-cell proliferation compared with the 2-day sham group; however, both sham and Px groups showed depressed β-cell proliferative activity at 2 days compared with untouched mice, likely due to suppressed feeding activity following laparotomy (Fig. 1D). Cyclin D2, a member of the D-type cyclin family, plays a crucial role in β-cell proliferation (4,5). We detected a marked increase in cyclin D2 protein by immunoblotting and immunoreactivity, with an increase in the number of β-cell nuclei expressing the protein in 6 days post-Px mice compared with sham mice (Fig. 1E). No differences were observed in cyclin D2 expression in the 15-day groups (data not shown).

BCM enhancement is associated with a transient increase in small β-cell clusters.

Although controversial, β-cell neogenesis has been proposed to play an important role in BCM compensation (10,17,2023). We therefore quantified the prevalence of β-cell clusters and islets and ranked them according to size to determine their contribution to BCM increase in C57BL/6 mice after Px. We observed an ∼2.8-fold higher incidence of β-cell clusters/small islets (1,000- to 4,000-μm2 range) in 2-day Px mice compared with sham controls (Fig. 1F). In contrast, we detected no increase in the number of very small β-cell clusters (one to four cells in cross-section) between the 2 and 6 day postsurgery groups at this time point (data not shown). Proliferation was also rare in both β-cell cluster sizes with no differences detected between groups. We next quantified the prevalence of islet sizes ranging ∼4,000–8,000, 8,000–11,960, 12,000–16,000, and >16,000 μm2 (Table 1). We detected an increase of about twofold in the larger 8,000–11,960 and 12,000–16,000 μm2 islet size groups at 15 days postsurgery (islet diameter range ∼90–125 μm) (Table 1). Collectively, these results seem consistent with an early postmitotic differentiation of β-cell precursors, which initially appear as small β-cell clusters and then subsequently proliferate to larger islets.

Enhanced BCM in 60% Px mice correlates with increased Akt signaling and PDX1 expression in pancreatic β-cells.

To determine if the IRS2/Akt/PDX1 signaling pathway is activated during the BCM increase, we analyzed the expression of these proteins at 6 days postsurgery when β-cell proliferation is maximal. By immunostaining, no differences were observed in β-cell IRS2 levels between groups (data not shown). Immunostaining and immunoblot analyses revealed low to moderate levels of pAkt in islet β-cells of control mice (Fig. 2A and C). However, surface-oriented pAkt immunoreactivity was enhanced in islets of 6-day Px mice (Fig. 2B and C). Furthermore, pAkt expression correlated with increased levels of PDX1 expression in β-cells (Fig. 2B and C).

Decreased FoxO1 but enhanced Foxa2 expression in regenerating β-cells.

The forkhead transcription factor FoxO1 plays key roles in regulating apoptosis, proliferation, and glucose metabolism. The function of FoxO1 is inhibited by Akt-mediated phosphorylation and its subsequent nuclear exclusion (24). Recent studies suggest that FoxO1 regulates BCM by inhibiting Pdx1 gene transcription through Foxa2 binding (12). We therefore examined FoxO1 and Foxa2 expression in mice at 2 and 6 days post-Px. Compared with sham mice, we observed a moderate decrease in total cytoplasmic FoxO1 by immunostaining and immunoblotting and a marked decrease in phospho-FoxO1 by immunoblotting in 6-day Px mice (Fig. 2D–F). Hence, it appears that both the total and phosphorylated (presumably cytoplasmic) pools of β-cell FoxO1 are reduced in 6-day Px mice. No changes were detected in strongly FoxO1+ peripheral cells that were often PDX1+/somatostatin+ (Fig. 2D and E).

We next analyzed the expression of Foxa2 in 6-day Px mice by immunostaining. We detected moderate increases in β-cell nuclear Foxa2 immunoreactivity compared with sham-operated mice (Fig. 2G and H).

Heightened proliferation in the CPD epithelium in the regenerating pancreas.

Our previous studies in the rat 60% Px model have shown that enhanced CPD proliferation was highest in the lateral evaginations as opposed to main epithelial cell lining (17). Accordingly, by immunostaining with Ki-67, we observed increased frequency of staining in the evaginations of 2-day Px mice (Fig. 3B and D) compared with shams (Fig. 3A and C). We quantified CPD proliferation, distinguishing between the main epithelial lining versus the evaginations. Compared with control mice, we detected a 10-fold increase in the mitotic frequency of the evaginations at 2 days post-Px (P < 0.001), which was sustained at 6 days post-Px (Fig. 3E). Although insulin+ cells in Px mice were associated with evaginations, their low frequency precluded a quantitative analysis.

Augmented insulin signaling in ducts of Px mice.

New β-cell development, or neogenesis, is widely considered to originate from the pancreatic exocrine ducts (17,20,2527). Hence, we next sought to examine the activity of the IRS2/Akt/FoxO1 pathway in the common pancreatic duct epithelium, since we detected transient increases in the number of small islets/β-cell clusters early in the regeneration period along with hyperproliferation of cells lining the duct evaginations. Our previous studies in the 60% Px rat model had demonstrated enhanced IRS2 and pAkt immunoreactivity in the CPD during pancreatic regeneration that correlated with β-cell neogenesis (17). Similarly, we also observed strong IRS2 staining in the CPD evaginations of 2- and 6-day Px mice that normalized by 15 days postsurgery (Fig. 4A and B). pAkt immunoreactivity was also increased at 2 and 6 days post-Px compared with sham ducts (Figs. 3A and B and 4C–E). Surface-oriented pAkt immunoreactivity correlated with increased nuclear PDX1 expression in Px mice, especially in the duct evaginations (Figs. 3D and 4D). Correspondingly, by immunoblotting, PDX1 protein levels were increased ∼1.5-fold in 2-day Px mice; however, pAkt levels were markedly enhanced at both 2 and 6 days post-Px in CPD extracts (Fig. 4E).

Since recent reports have demonstrated FoxO1 expression in pancreatic ducts and because it antagonizes Foxa2 function in islets (12), we first examined FoxO1 and Foxa2 mRNA by RT-PCR in ducts and islets of Px rats. An increase of ∼1.5-fold in FoxO1 and Foxa2 mRNA levels over sham controls was observed in isolated CPDs from 2-day Px rats, while there was a marginal but significant decrease in islets (Fig. 5A and B). By immunostaining and immunoblotting, intense cytoplasmic FoxO1 (Fig. 5C, D, and G) and nuclear Foxa2 immunoreactivity (Fig. 5E and F) were observed in the regenerating duct epithelium, especially in the evaginations. The contrasting profiles of FoxO1 mRNA and protein levels in islets and ducts suggest different regulatory mechanisms in these tissues.

In vivo insulin administration to Px mice augments β-cell Akt signaling.

To compare the status of the insulin signaling intermediates between a chronic (i.e., Px surgery) and short-term stimulus, insulin (750 mU/kg i.p.) or saline was administered to 6-day sham and Px mice and then analyzed at intervals for IRS2/pAkt/FoxO1 and PDX1 immunoreactivity in islets and ducts. At 10 min postinjection, although increased surface-oriented pAkt was observed in β-cells of saline-treated Px mice compared with shams (Fig. 6A and B), there was a striking enhancement in cytoplasmic pAkt in insulin-treated Px β-cells compared with sham islets (Fig. 6C and D). Unexpectedly, we also observed enhanced IRS2 immunoreactivity in the islets of insulin-treated Px mice compared with shams (Fig. 6E and F). FoxO1 immunoreactivity was drastically reduced in insulin-treated Px mice compared with insulin-treated sham mice (Fig. 6G and H). In contrast, increased FoxO1 immunoreactivity was observed in the CPD evaginations of insulin-treated Px mice compared with a corresponding treatment of control mice (Fig. 6I and J). These results show that the observed pattern of tissue-specific signaling intermediate expression and activation noted post-Px was augmented by exogeneous insulin.

Although the metabolic characteristics of compensatory partial regeneration of the pancreatic β-cells have been well characterized in the Px rodent model (17,28,29), a detailed study of the underlying mechanisms that are responsible for the BCM expansion have not been previously reported. Previous studies in C57BL/6 (18) and other mouse strains (3032) following Px have highlighted the impact of β-cell proliferation in BCM homeostasis. On the other hand, pancreatic duct ligation (33) and glucose-infusion rodent models (34) have emphasized BCM contributions by neogenesis originating from exocrine tissues. However, the confounding effects of hyperglycemia in these models on BCM compensation, the signaling pathways, and β-cell gene expression could not be excluded (2). In contrast, the 60% Px rodent model offers a significant advantage in examining BCM compensation since a vigorous, but transient, regeneration process ensues despite a 60% reduction in BCM (17,18).

In this study, we have investigated the mechanisms that mediate BCM expansion in 60% Px C57BL/6 mice. There was a net increase in BCM of ∼28% versus shams by 15 days following Px due to an increase in the number of β-cell clusters/small islets, possibly from an early postmitotic differentiation of as yet unidentified β-cell precursors, followed by enhanced β-cell proliferation in preexisting islets. We have demonstrated that these consecutive processes correlate strongly with enhanced insulin receptor pathway signaling, with Akt kinase serving as a central intermediate.

The role of insulin signaling–mediated duct proliferation and differentiation in BCM compensation.

Previous studies in both 60 and 90% Px rat models have shown that proliferation preceded differentiation in the epithelium of the large ducts (17,28). We detected a massive 10-fold increase in proliferation in the epithelial lining of the lateral evaginations of the CPD at 2 days post-Px. Concomitant with an increase in proliferation, we observed a transient activation of the insulin signaling pathway in the CPD, as evidenced by increased IRS2, pAkt, and FoxO1 immunoreactivity in the C57BL/6 mice post-Px in concert with enhanced PDX1 expression. Thus, in 2-day post-Px mice, PDX1, IRS2, activated Akt, FoxO1, and Foxa2 were all expressed in the CPD lining; however, they were more robustly expressed in the epithelial cells of the duct evaginations versus the lining. More importantly, strong pAkt+ cells correlated with intense nuclear PDX1 expression. However, since very few insulin+ cells were detected within the ducts of the sham and Px mice, it is unclear whether these strong pAkt+/PDX1+ cells progress to fully differentiated β-cells to contribute to the islet β-cell pool.

In parallel with a peak in duct proliferation and enhanced Akt signaling at 2 days post-Px, there was a short-lived 2.8-fold increase in the number of β-cell clusters and small islets. As the prevalence of these clusters waned, β-cell proliferation peaked at 6 days post-Px, correlating with a 50% increase in BCM. By 15 days postsurgery, increased numbers of large islets were observed in Px mice, suggesting continued growth of the islet β-cell population following the transient β-cell hyperproliferation occurring 1 week prior. Although there are currently no specific markers for newly differentiated versus newly replicated β-cells, these results suggest that in this mouse model of Px, BCM expansion involves not only the expected proliferation from preexisting β-cells but possibly from newly formed islets. The contribution of β-cell neogenesis from ducts or other pancreatic progenitor cells to both normal BCM homeostasis (1,35) and to an experimental regeneration stimulus (17,25,34) has recently been challenged by studies reporting that the primary mechanism of β-cell renewal is by proliferation of preexisting β-cells (3). Although our results strongly suggest that the early increase in the number of β-cell clusters likely contributes to BCM compensatory increase, the source of these β-cell clusters/small islets remains unknown, as we failed to detect proliferating β-cell clusters, and the frequency of insulin+ cells in the duct epithelium was too low to be quantified. However, increased numbers of single/double β-cells have been observed in transgenic mice overexpressing constitutively active Akt1 (9). Thus, in the current study, BCM expansion due to Akt activation may not only regulate hyperproliferation of existing β-cells but, possibly, β-cell differentiation from exocrine progenitors as well.

Role of proliferation during BCM homeostasis.

Studies of mice with altered gene expression suggest that the insulin signaling pathway via Akt/FoxO1/PDX1 regulates β-cell proliferation (7,12,1416). In turn, the β-cell proliferative response is associated with increased levels of cell cycle proteins, including cyclin D1, cyclin D2, p21, and cdk4 activity mediated by Akt (4,5,36,37). Thus, whereas FoxO1 haplodeficiency restored Pdx1 expression and β-cell proliferation in IRS2−/− and Pdk1−/− mice (12,15), PDX1 haplodeficiency abrogated β-cell compensatory response in insulin-resistant Insr+/−/Irs1+/− mice and in mice lacking insulin receptor in liver through impaired β-cell–proliferation mice (14). These results suggest that FoxO1 and PDX1 may mediate proliferative signals through Akt.

A universal finding in normal rodent Px models is the several-fold increase in β-cell proliferation peaking 4–7 days postsurgery. Individual islet growth, including that following a Px stimulus, would be expected to be a product of β-cell proliferation, hypertrophy, apoptosis, and clearance (1,17,38). This was suggested by a 2.5-fold enhancement in β-cell proliferation at 6 days post-Px, concomitant with increased cyclin D2 expression. Thus, enhanced Akt signaling in β-cells from 6 days post-Px mice likely mediates this heightened replication.

Insulin signaling mediates BCM homeostasis in Px mice.

Recent studies indicate that the IRS2/Akt/FoxO1 signaling pathway plays a pivotal role in compensatory BCM augmentation in insulin-resistance states, with β-cell proliferation as a primary mechanism for maintenance of this BCM (716,39). Rescue of diabetes in Pdk1−/− and Irs2−/− mice by superimposing FoxO1 haploinsufficiency, and by over-expression of PDX1 in Irs2−/− mice, essentially restored BCM and PDX1 expression, implying a regulatory link between the insulin signaling cascade and PDX1 (12,13,15). Although the impact of this pathway in response to β-cell regeneration is not yet fully resolved, we have previously reported in the 60% rat Px model that increased IRS2 and pAkt levels correlated with enhanced neogenesis from the large ducts (17). While IRS2 levels in β-cells were not significantly changed during β-cell regeneration, possibly due to degradation (40), β-cell pAkt immunoreactivity was intense and surface oriented and, at 6 days post-Px, correlated strongly with nuclear PDX1. Akt has been ascribed roles in regulating β-cell size, survival, proliferation (911,41,42), and neogenesis (9,17). Many of these functions are likely manifestations of Akt signaling through the transcription factor FoxO1 and, in turn, PDX1. In islets of Px mice, cytoplasmic FoxO1 expression was decreased profoundly in β-cells of 6-day Px mice and correlated with enhanced PDX1 levels. Although not substantiated, these observations suggest that phosphorylated FoxO1 may be rapidly degraded following nuclear exclusion and PDX1 activation (12,43). In contrast to islets, increased cytoplasmic expression of FoxO1 correlated with increased nuclear PDX1 expression in the CPD evaginations. This observation was puzzling, since we anticipated decreased cytoplasmic FoxO1 immunoreactivity due to intense nuclear PDX1 and Foxa2 immunoreactivity in the CPD evaginations. Although unclear, the regulation of FoxO1 activity appears tissue specific and warrants further investigation.

We have also detected moderate increases in Foxa2 mRNA and protein levels in Px mice consistent with its role as an activator of PDX1 expression (44). As PDX1 has recently been ascribed roles in β-cell survival and proliferation (14,45), these results support our premise that enhanced BCM in Px mice involves β-cell signaling through Akt, FoxO1, and PDX1, regulating β-cell growth. Our findings that a transient stimulation of the insulin signaling pathway by insulin itself further enhanced the expression pattern of signaling elements during β-cell regeneration underscores the impact of this pathway in BCM expansion.

In conclusion, the 60% Px model serves as a useful paradigm for examining the mechanisms and signaling pathways for β-cell regeneration following pancreatic injury. The current study in the C57BL/6 mouse strain demonstrates that β-cell regeneration entails coordinated processes that involve an early increase in the prevalence of β-cell clusters with no demonstrable proliferation of these insulin+ cells. Subsequently, β-cell hyperproliferation occurs as a second-phase response to the β-cell regeneration stimulus. The IRS2/Akt/FoxO1/PDX1 pathway appears to mediate this β-cell growth response. We speculate that an early response involving increased insulin signaling activity and proliferation in pancreatic ducts post-Px may be related to neogenesis.

FIG. 1.

Metabolic and β-cell growth characteristics in 60% Px mice. Fed blood glucose (A) and insulin concentrations (B) were comparable between sham and Px mice through 15 days (d) postsurgery (n ≥ 10). Values are means ± SE. C: BCM increased progressively during the 2 weeks postsurgery in Px mice compared with sham controls. Untouched 2- and 15-day mice are shown for comparison. The black line superimposed on the sham bars represents the theoretical BCM postsurgery. *P < 0.01; **P < 0.001; n > 5. D: Compared with shams, β-cell proliferation increased 2.54-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 image and immunoblot showing increased cyclin D2 signal in 6-day Px islet compared with sham controls. F: A 2.8-fold increase in β-cell clusters and small islets was observed in 2-day Px mice compared with sham animals. *P < 0.05.

FIG. 1.

Metabolic and β-cell growth characteristics in 60% Px mice. Fed blood glucose (A) and insulin concentrations (B) were comparable between sham and Px mice through 15 days (d) postsurgery (n ≥ 10). Values are means ± SE. C: BCM increased progressively during the 2 weeks postsurgery in Px mice compared with sham controls. Untouched 2- and 15-day mice are shown for comparison. The black line superimposed on the sham bars represents the theoretical BCM postsurgery. *P < 0.01; **P < 0.001; n > 5. D: Compared with shams, β-cell proliferation increased 2.54-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 image and immunoblot showing increased cyclin D2 signal in 6-day Px islet compared with sham controls. F: A 2.8-fold increase in β-cell clusters and small islets was observed in 2-day Px mice compared with sham animals. *P < 0.05.

FIG. 2.

Enhanced pAkt, PDX1, and Foxa2, but decreased FoxO1, expression in Px mouse islets. A and B: Surface pAkt and nuclear PDX1 immunoreactivity were increased in Px islets compared with low levels detected in 6-day sham islets. C: Islets from representative 6-day animals were 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 β-cells compared with sham islets. Peripheral PDX1+δ 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 antibody followed by total FoxO1 antibody. G and H: A wide-field immunofluorescence image of representative islets showing modest increases in Foxa2 in 6-day Px islet compared with shams.

FIG. 2.

Enhanced pAkt, PDX1, and Foxa2, but decreased FoxO1, expression in Px mouse islets. A and B: Surface pAkt and nuclear PDX1 immunoreactivity were increased in Px islets compared with low levels detected in 6-day sham islets. C: Islets from representative 6-day animals were 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 β-cells compared with sham islets. Peripheral PDX1+δ 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 antibody followed by total FoxO1 antibody. G and H: A wide-field immunofluorescence image of representative islets showing modest increases in Foxa2 in 6-day Px islet compared with shams.

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 were observed in the sham duct lining and evaginations (arrow). B: A striking increase in Ki-67+/pAkt+ cells, concentrated in the evaginations (arrows), was observed in the Px ducts. C: High magnification field of an evagination (arrow) in shams with little mitotic activity, as opposed to several PDX1+/Ki-67+ cells (D) in evaginations of Px mice (arrow). AD: *Duct lumen. E: In contrast to common duct epithelial lining, proliferation frequency in the 2-day Px animals, compared with shams, is substantially increased in evaginations (*P < 0.001). A moderate increase was observed in 6-day Px evaginations, which normalized by 15 days.

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 were observed in the sham duct lining and evaginations (arrow). B: A striking increase in Ki-67+/pAkt+ cells, concentrated in the evaginations (arrows), was observed in the Px ducts. C: High magnification field of an evagination (arrow) in shams with little mitotic activity, as opposed to several PDX1+/Ki-67+ cells (D) in evaginations of Px mice (arrow). AD: *Duct lumen. E: In contrast to common duct epithelial lining, proliferation frequency in the 2-day Px animals, compared with shams, is substantially increased in evaginations (*P < 0.001). A moderate increase was observed in 6-day Px evaginations, which normalized by 15 days.

FIG. 4.

Increased insulin signaling activity and PDX1 expression in 2-day Px mouse duct evaginations. Although low levels of IRS2 and phosphotyrosine (pY) (A) were detected in sham duct lining and evaginations (arrow), increased IRS2 levels (B), with frequently more pY+ 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 and PDX1 staining (arrows). AD: *Duct lumen. E: A representative immunoblot of 2- and 6-day sham and Px ducts probed with PDX1, pAkt, and total Akt antibodies. β-Catenin was used as a loading control.

FIG. 4.

Increased insulin signaling activity and PDX1 expression in 2-day Px mouse duct evaginations. Although low levels of IRS2 and phosphotyrosine (pY) (A) were detected in sham duct lining and evaginations (arrow), increased IRS2 levels (B), with frequently more pY+ 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 and PDX1 staining (arrows). AD: *Duct lumen. E: A representative immunoblot of 2- and 6-day sham and Px ducts probed with PDX1, pAkt, and total Akt antibodies. β-Catenin was used as a loading control.

FIG. 5.

Enhanced FoxO1 and Foxa2 in mouse Px duct evaginations. A: Semiquantitative RT-PCR analysis showing increased FoxO1 mRNA levels in 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 comparable field from a Px mouse displaying heightened FoxO1 staining in evaginations. E: Moderate levels of nuclear Foxa2 staining were observed in the main 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 with a further enhancement in evaginations (arrows). CF: *Lumen. G: Ducts from representative 2- and 6-day sham and Px mice were probed with pFoxO1 antibody. Actin served as a loading control.

FIG. 5.

Enhanced FoxO1 and Foxa2 in mouse Px duct evaginations. A: Semiquantitative RT-PCR analysis showing increased FoxO1 mRNA levels in 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 comparable field from a Px mouse displaying heightened FoxO1 staining in evaginations. E: Moderate levels of nuclear Foxa2 staining were observed in the main 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 with a further enhancement in evaginations (arrows). CF: *Lumen. G: Ducts from representative 2- and 6-day sham and Px mice were probed with pFoxO1 antibody. Actin served as a loading control.

FIG. 6.

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

FIG. 6.

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

TABLE 1

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

Prevalence/cm2 of medium to large size class islets
4,000–8,0008,000–11,96012,000–16,000>16,000
2-day sham 0.063 ± 0.012 0.025 ± 0.007 0.026 ± 0.008 0.015 ± 0.007 
2-day Px 0.085 ± 0.027 0.031 ± 0.013 0.031 ± 0.007 0.010 ± 0.005 
6-day sham 0.069 ± 0.010 0.030 ± 0.005 0.030 ± 0.005 0.016 ± 0.004 
6-day Px 0.085 ± 0.019 0.035 ± 0.007 0.039 ± 0.011 0.026 ± 0.009 
15-day sham 0.096 ± 0.012 0.029 ± 0.006 0.028 ± 0.006 0.016 ± 0.004 
15-day Px 0.113 ± 0.019 0.059 ± 0.010* 0.055 ± 0.011* 0.037 ± 0.013 
Prevalence/cm2 of medium to large size class islets
4,000–8,0008,000–11,96012,000–16,000>16,000
2-day sham 0.063 ± 0.012 0.025 ± 0.007 0.026 ± 0.008 0.015 ± 0.007 
2-day Px 0.085 ± 0.027 0.031 ± 0.013 0.031 ± 0.007 0.010 ± 0.005 
6-day sham 0.069 ± 0.010 0.030 ± 0.005 0.030 ± 0.005 0.016 ± 0.004 
6-day Px 0.085 ± 0.019 0.035 ± 0.007 0.039 ± 0.011 0.026 ± 0.009 
15-day sham 0.096 ± 0.012 0.029 ± 0.006 0.028 ± 0.006 0.016 ± 0.004 
15-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.

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.

This work was supported by a CDA from the Juvenile Diabetes Research Foundation and a Research Award from the American Diabetes Association (to M.P.) and by the 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 critical reading of the manuscript. We also are indebted to Dr. Afshin Salsali who helped in the initiation of this project.

1.
Bonner-Weir S: Islet growth and development in the adult.
J Mol Endocrinol
24
:
297
–302,
2000
2.
Jonas JC, Sharma A, Hasenkamp W, Ilkova H, Patane G, Laybutt R, Bonner-Weir S, Weir GC: Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
J Biol Chem
274
:
14112
–14121,
1999
3.
Dor Y, Brown J, Martinez OI, Melton DA: Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.
Nature
429
:
41
–46,
2004
4.
Georgia S, Bhushan A: Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass.
J Clin Invest
114
:
963
–968,
2004
5.
Kushner JA, Ciemerych MA, Sicinska E, Wartschow LM, Teta M, Long SY, Sicinski P, White MF: Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth.
Mol Cell Biol
25
:
3752
–3762,
2005
6.
Teta M, Long SY, Wartschow LM, Rankin MM, Kushner JA: Very slow turnover of β-cells in aged adult mice.
Diabetes
54
:
2557
–2567,
2005
7.
Withers D, Gutierrez JS, Towery H, Burks DJ, Ren JM, Previs S, Zhang Y, Bernal D, Pons S, Shulman GI, Bonner-Weir S, White MF: Disruption of IRS-2 causes type 2 diabetes in mice.
Nature
391
:
900
–904,
1998
8.
Kubota N, Tobe K, Terauchi Y, Eto K, Yamauchi T, Suzuki R, Tsubamoto Y, Komeda K, Nakano R, Miki H, Satoh S, Sekihara H, Sciacchitano S, Lesniak M, Aizawa S, Nagai R, Kimura S, Akanuma Y, Taylor SI, Kadowaki T: Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory β-cell hyperplasia.
Diabetes
49
:
1880
–1889,
2000
9.
Bernal-Mizrachi E, Wen W, Stahlhut S, Welling CM, Permutt MA: Islet beta cell expression of constitutively active Akt1/PKB alpha induces striking hypertrophy, hyperplasia, and hyperinsulinemia.
J Clin Invest
108
:
1631
–1638,
2001
10.
Tuttle RL, Gill NS, Pugh W, Lee JP, Koeberlein B, Furth EE, Polonsky KS, Naji A, Birnbaum MJ: Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha.
Nat Med
7
:
1133
–1137,
2001
11.
Garofalo RS, Orena SJ, Rafidi K, Torchia AJ, Stock JL, Hildebrandt AL, Coskran T, Black SC, Brees DJ, Wicks JR, McNeish JD, Coleman KG: 2003: severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta.
J Clin Invest
112
:
197
–208,
2003
12.
Kitamura T, Nakae J, Kitamura Y, Kido Y, Biggs WH, Wright CV, White MF, Arden KC, Accili D: The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growth.
J Clin Invest
110
:
1839
–1847,
2002
13.
Kushner JA, Ye J, Schubert M, Burks DJ, Dow MA, Flint CL, Dutta S, Wright CV, Montminy MR, White MF: Pdx1 restores beta cell function in Irs2 knockout mice.
J Clin Invest
109
:
1193
–1201,
2002
14.
Kulkarni RN, Jhala US, Winnay JN, Krajewski S, Montminy M, Kahn CR: PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance.
J Clin Invest
114
:
828
–836,
2004
15.
Hashimoto N, Kido Y, Uchida T, Asahara S, Shigeyama Y, Matsuda T, Takeda A, Tsuchihashi D, Nishizawa A, Ogawa W, Fujimoto Y, Okamura H, Arden KC, Herrera PL, Noda T, Kasuga M: Ablation of PDK1 in pancreatic beta cells induces diabetes as a result of loss of beta cell mass.
Nat Genet
38
:
589
–593,
2006
16.
Ueki K, Okada T, Hu J, Liew CW, Assmann A, Dahlgren GM, Peters JL, Shackman JG, Zhang M, Artner I, Satin LS, Stein R, Holzenberger M, Kennedy RT, Kahn CR, Kulkarni RN: Total insulin and IGF-I resistance in pancreatic beta cells causes overt diabetes.
Nat Genet
38
:
583
–588,
2006
17.
Jetton TL, Liu YQ, Trotman WE, Nevin PW, Sun XJ, Leahy JL: Enhanced expression of insulin receptor substrate-2 and activation of protein kinase B/Akt in regenerating pancreatic duct epithelium of 60%-partial pancreatectomy rats.
Diabetologia
44
:
2056
–2065,
2001
18.
Martin F, Andreu E, Rovira JM, Pertusa JA, Raurell M, Ripoll C, Sanchez-Andres JV, Montanya E, Soria B: Mechanisms of glucose hypersensitivity in β-cells from normoglycemic, partially pancreatectomized mice.
Diabetes
48
:
1954
–1961,
1999
19.
Sutton R, Peters M, McShane P, Gray DW, Morris PJ: Isolation of rat pancreatic islets by ductal injection of collagenase.
Transplantation
42
:
689
–691,
1986
20.
Sharma A, Zangen DH, Reitz P, Taneja M, Lissauer ME, Miller CP, Weir GC, Habener JF, Bonner-Weir S: The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration.
Diabetes
48
:
507
–513,
1999
21.
Jetton TL, Lausier J, LaRock K, Trotman WE, Larmie B, Habibovic A, Peshavaria M, Leahy JL: Mechanisms of compensatory β-cell growth in insulin-resistant rats: roles of Akt kinase.
Diabetes
54
:
2294
–2304,
2005
22.
Risbud MV, Bhonde RR: Models of pancreatic regeneration in diabetes.
Diabetes Res Clin Pract
58
:
155
–165,
2002
23.
Meier JJ, Bhushan A, Butler AE, Rizza RA, Butler PC: Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration?
Diabetologia
48
:
2221
–2228,
2005
24.
Czech MP: Insulin’s expanding control of forkheads.
Proc Natl Acad Sci U S A
100
:
11198
–11200,
2003
25.
Woodroof CW, de Villiers C, Page BJ, van der Merwe L, Ferris WF: Islet neogenesis is stimulated by brief occlusion of the main pancreatic duct.
S Afr Med J
94
:
54
–57,
2004
26.
Gu D, Sarvetnick N: Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice.
Development
118
:
33
–46,
1993
27.
Paris M, Tourrel-Cuzin C, Plachot C, Ktorza A: Review: pancreatic beta-cell neogenesis revisited.
Exp Diabesity Res
5
:
111
–121,
2004
28.
Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE: A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development.
Diabetes
42
:
1715
–1720,
1993
29.
Lee HC, Bonner-Weir S, Weir GC, Leahy JL: Compensatory adaption to partial pancreatectomy in the rat.
Endocrinology
124
:
1571
–1575,
1989
30.
Hardikar AA, Karandikar MS, Bhonde RR: Effect of partial pancreatectomy on diabetic status in BALB/c mice.
J Endocrinol
162
:
189
–195,
1999
31.
Anneren C: Dual role of the tyrosine kinase GTK and the adaptor protein SHB in beta-cell growth: enhanced beta-cell replication after 60% pancreatectomy and increased sensitivity to streptozotocin.
J Endocrinol
172
:
145
–153,
2002
32.
Lee CS, De Leon DD, Kaestner KH, Stoffers DA: Regeneration of pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3.
Diabetes
55
:
269
–272,
2005
33.
Wang RN, Kloppel G, Bouwens L: Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats.
Diabetologia
38
:
1405
–1411,
1995
34.
Lipsett M, Finegood DT: β-Cell neogenesis during prolonged hyperglycemia in rats.
Diabetes
51
:
1834
–1841,
2002
35.
Bernard-Kargar C, Ktorza A: Endocrine pancreas plasticity under physiological and pathological conditions.
Diabetes
50 (Suppl. 1)
:
S30
–S35,
2001
36.
Fatrai S, Elghazi L, Balcazar N, Cras-Meneur C, Krits I, Kiyokawa H, Bernal-Mizrachi E: Akt induces β-cell proliferation by regulating cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 activity.
Diabetes
55
:
318
–325,
2006
37.
Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, Barbacid M: Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.
Nat Genet
22
:
44
–52,
1999
38.
Scaglia L, Cahill CJ, Finegood DT, Bonner-Weir S: Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal rat.
Endocrinology
138
:
1736
–1741,
1997
39.
Nakae J, Biggs WH, Kitamura T, Cavenee WK, Wright CV, Arden KC, Accili D: Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor FoxO1.
Nat Genet
32
:
245
–253,
2002
40.
Briaud I, Dickson LM, Lingohr MK, McCuaig JF, Lawrence JC, Rhodes CJ: Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells.
J Biol Chem
280
:
2282
–2293,
2005
41.
Elghazi L, Balcazar N, Bernal-Mizrachi E: Emerging role of protein kinase B/Akt signaling in pancreatic beta-cell mass and function.
Int J Biochem Cell Biol
38
:
157
–163,
2006
42.
Dickson LM, Rhodes CJ: Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt?
Am J Physiol Endocrinol Metab
287
:
E192
–E198,
2004
43.
Aoki M, Jiang H, Vogt PK: Proteasomal degradation of the FoxO1 transcriptional regulator in cells transformed by the P3k and Akt oncoproteins.
Proc Natl Acad Sci U S A
101
:
13613
–13617,
2004
44.
Wu KL, Gannon M, Peshavaria M, Offield MF, Henderson E, Ray M, Marks A, Gamer LW, Wright CV, Stein R: Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.
Mol Cell Biol
17
:
6002
–6013,
1997
45.
Johnson JD, Ahmed NT, Luciani DS, Han Z, Tran H, Fujita J, Misler S, Edlund H, Polonsky KS: Increased islet apoptosis in Pdx1+/- mice.
J Clin Invest
111
:
1147
–1160,
2003