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Am J Physiol Gastrointest Liver Physiol 294: G1139-G1147, 2008. First published March 27, 2008; doi:10.1152/ajpgi.00338.2007
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HORMONES AND SIGNALING

Selective expansion of the β-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Martin Wagner,1 Stefan Koschnick,2 Sven Beilke,1 Melanie Frey,1 Guido Adler,1 and Roland M. Schmid3

1Department of Internal Medicine I, University Ulm; 2Herzzentrum University, Göttingen; and 3Department of Internal Medicine II, Technische Universität München

Submitted 25 July 2007 ; accepted in final form 16 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the β-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of β-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the β-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the β-cell compartment does not necessarily result in an improved glucose tolerance in vivo.

β-cell proliferation; protein kinase B; glucose tolerance

THE PANCREAS CONSISTS OF EXOCRINE tissue and endocrine cells, which are arranged in the islets of Langerhans. During development, pluripotent precursor cells differentiate into either exocrine or endocrine tissue. The transition to mature islets requires an orderly genetic program for further differentiation into endocrine cells and islets with a sophisticated intraislet portal system. This process is strongly dependent on mesenchymal-epithelial interactions (11, 18, 19). The mature pancreatic islets consist of β-cells secreting insulin, {alpha}-cells secreting glucagon, {delta}-cells secreting somatostatin, and PP cells secreting pancreatic polypeptide (16).

Insulin-producing β-cells develop through neogenesis from precursor cells and/or self-replication. The entire compartment of β-cells in the adult is established in the prenatal period, and after birth the rate of cell division is quite low with a mitotic activity of ~0.5% primarily by replication of differentiated cells (9). Under certain conditions, the capacity of β-cell regeneration is evident (4, 10, 18, 26). The endocrine tissue displays a greater regenerative capacity after duct ligation as well as after partial pancreatectomy compared with the exocrine part of the organ (4, 35). Overexpression of interferon-{gamma} (IFN-{gamma}) in transgenic mice using the rat proinsulin promoter leads to the development of clusters of endocrine cells within the wall of proliferating ducts (13). This phenotype is associated with an upregulation of epidermal growth factor, transforming growth factor-{alpha}, the epidermal growth factor receptor, and keratinocyte growth factor (KGF) (1, 15). These findings have strengthened the concept that β-cells differentiate from duct cells or a stem cell population within this compartment. However, a more recent publication demonstrates the self-renewal capacity of mature β-cells. In linage tracing experiments, new β-cells seem to arise from self-duplication rather than from pancreatic ducts or a so far undefined stem cell compartment (5).

KGF, also known as fibroblast growth factor-7, is a secreted, mesenchymal-derived growth factor acting as a paracrine effector on epithelial proliferation and differentiation through the interaction with a splice variant of the fibroblast growth factor receptor 2 (FGFR2IIIb) (8, 23). Whereas KGF is produced and secreted exclusively by mesenchymal-derived cells, the KGF receptor is expressed on epithelial cells. KGF plays a major role in mediating mesenchymal-epithelial interaction during fetal development and regenerative processes in adults (18, 36, 37). In particular, KGF is capable of inducing expansion of human fetal β-cells after transplantation in vivo (20). KGF represents a major component in differentiation medium of embryonic stem cells toward insulin-producing cells (e.g., Ref. 3), and KGF-dependent signals are essential for the expansion of endocrine progenitor cells from embryonic pancreatic epithelium in vitro (7).

KGF signaling events downstream of the FGFR2IIIb receptor tyrosine kinase include the activation of the protein kinase B (PKB)/Akt pathway in vivo. This activation promotes cell survival mechanisms in a model system of lung injury (24, 25, 27). The effects of the PKB/Akt pathway on the endocrine compartment of the pancreas are well described in several in vitro and in vivo models (17). Along these lines, mice overexpressing a constitutively active form of the protein kinase Akt1 (PKB-{alpha}) in β-cells have been shown to develop β-cell hypertrophy, hyperplasia, and increased insulin secretion (2, 30). A comparable phenotype is observed in animals with β-cell-specific deletion of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), the negative regulator of the Akt pathway. As a consequence of PTEN inactivation, Akt serine/threonine kinase and its downstream effectors are activated (21, 29). These results imply the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and upstream activators of this pathway as potential therapeutic targets for β-cell protection and regeneration.

Here we provide evidence that the ectopic expression of KGF in the exocrine compartment activates the Akt pathway in pancreatic islets in vivo, resulting in an expansion of the insulin-expressing cells. Interestingly, the functional analysis reveals defects in glucose homeostasis and insulin secretion in these KGF transgenic mice.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation and characterization of transgenic mice. A 0.6-kb XhoI/HindIII fragment including the cDNA of the murine KGF was subcloned into the E0.2-hGH plasmid carrying the rat elastase promoter (–205 to +5) and a genomic fragment of the human growth hormone (hGH) gene. In this construct the hGH sequences serve as 5' untranslated region and poly-A+ signal as previously described (22). Transgenic mice were generated by pronuclear injection of a purified 3.5-kb fragment into Cl57Bl/6J/DBA/2J F2 hybrid zygotes by standard procedures (34). This construct was used successfully to study the paracrine effects of growth factors on pancreatic differentiation in several previous publications (22, 33, 34). The genotype was screened by PCR analysis (FP1 5'-GGC TTT TTG ACA ACG CTA TG, RP1 5'-GCG CGG AGC ATA GGG TTG TC, FP2 5'-AAG AGC CGT ATA AAG AGG GT, and RP2 5'-GAT TTC CAT GAT GTT GTA GC). Animals were kept under specific pathogen-free conditions. Standard diet and water was supplied ad libitum. Transgenic lines were established from two independent founders, crossed to the DBA/2OlaHsd (Harlan, Oxon, UK) background, and analyzed after six to nine backcrosses. Blood glucose was measured in whole blood from tail vein (Glucometer Elite; Bayer, Leverkusen, Germany), and glucose tolerance test was performed after a 24-h fast (injection of 2 mg dextrose/g body wt in sterile PBS ip). Insulin levels were measured by radioimmunoassay (Insulin RIA; Linco, St. Charles, MI) following the manufactures recommendation in selected animals euthanized at the given time point. Furthermore, body weight, pancreatic wet weight, and the relation of body weight to pancreatic weight were measured during these experiments. All experiments were approved by the University of Ulm Animal Care and Use Committee and complied with the guidelines set by the National Institutes of Health.

Quantitative RT-PCR and in situ RNA hybridization. Total RNA was extracted as previously described. Transgene expression was evaluated by using quantitative RT-PCR (TaqMan assay; PE Applied Biosystems, Norwalk, CT) as described (31). Primers (5'-TGA CAC CTA CCA GGA GTT TGA AGA AG, 5'-ATT TCT GTT GTG TTT CCT CCC TGT T, and the FAM-labeled internal probe 5'-FAM-CCC TCT GTT TCT CAG AGT CTA TTC CGA CAC CC) specifically recognized the spliced hGH sequence at the 3' end of the transgene and did not react with endogenous KGF. RT-PCR was carried out in triplicate and was normalized to endogenous 18S mRNA levels (Ribosomal control reagent, PE Applied Biosystems) resulting in a {Delta}{Delta}Ct value with {Delta}Ct representing the difference between the threshold cycle of the target cDNA and the internal control (18S mRNA). Using this approach, low {Delta}Ct values indicate high expression and vice versa. Specific amplification was confirmed by electrophoresis on a 4% low melting agarose gel. Correlation between the morphometric analysis and the expression of the transgene was established with Pearson multiple regression using Statistica (StatSoft, Tulsa, OK).

Quantification of mRNA expression of insulin, Pdx1, and glucagon was performed with the Sybr green reagent, normalized to endogenous cyclophilin expression, and expression of the target mRNA was quantified using the {Delta}{Delta}Ct method as reported. Primer sequences are available on request (34).

Furthermore, expression of the FGFR2IIIb and IIIc splice variants was evaluated with RT-PCR of laser captured microdissected pancreatic tissue as described (34). In short, total RNA was isolated from microdissected material, reverse transcribed, and amplified with PCR. In a nested PCR reaction primers FP1 (5'-CTGCAAGGTTTACAGCGATGC) and RP1 (5'-ATAGCTATCTCCAGATAATCTGG) were used to amplify the common part of FGFR2 followed by the exon-specific amplification of FGFR2IIIc (FP2, 5'-ATCCAGTGGATCAAGCACGTG; RP4, 5'-AAAGGATATCCCGATAGAATTAC; PCR product 196 bp) and FGFR2IIIb (FP2, 5'-ATCCAGTGGATCAAGCACGTG; RP5, 5'-CTGCCCTATATAATTGGAGACC; PCR product 183 bp). RNA of whole embryo e15.5 served as a control.

The transgene expression was confirmed by nonradioactive RNA in situ hybridization as previously described (28, 34) with the spliced hGH as template for digoxigenin-labeled antisense and sense RNA probe. The hGH probe represents the spliced 5' region of the construct. This allows specific detection of the transgene-derived mRNA. Hybridization signal is represented by the blue staining (alkaline phosphatase/NBT-BCIP), and the nuclei were counterstained with methylene green.

Light microscopy and morphometric evaluation. Pancreatic tissue was fixed in 4% PBS buffered paraformaldehyde solution for 12 h, embedded in paraffin, sectioned (4 µm), and stained with hematoxylin/eosin. The pancreas was embedded together with the spleen under light pressure to achieve proper orientation and plain sections through the whole organ. For morphometric analysis, five sections separated by at least 100 µm were stained either with hematoxylin/eosin or immunostained for insulin or glucagon (see below) and examined at a magnification of x25. The whole section was digitalized using AMBA/W (version 3.0A; IBSB, Berlin, Germany). The number of islets, the islet area, as well as the area of exocrine tissue were evaluated and expressed as arbitrary unit areas. Single endocrine cells were excluded from the analysis of the number of islets. Values are expressed as mean of numbers determined from five sections. Six transgenic animals and six littermates were analyzed. For statistical analysis the Mann-Whitney-Wilcoxon test was used. P < 0.05 was considered significant.

Immunohistochemistry and Western blot analysis. Immunohistochemical analysis was performed on paraffin sections for transgenic animals and littermate controls as previously described (34, 32). The following primary antibodies were used: anti-Nkx2.2 (1:50; clone 74.5A5) anti-islet 1 (1:50; clone 39.4D5, both developmental studies hybridoma bank), anti-Pax6 (1:50; clone PAX6, developmental studies hybridoma bank), anti-insulin (1:200, Linco), anti-amylin (1:100, SC20936; Santa Cruz Biotechnology, Santa Cruz, CA), anti-glucagon (1:200; DAKO, Glostrup, Denmark), anti-Ki67 (1:100, DAKO), anti-murine FGF type 2 receptor (1:100, Santa Cruz Biotechnology), anti-pancreatic polypeptide (1:100; Binding Site, Birmingham, UK), anti-somatostatin (1:2,000; Biotrend, Cologne, Germany), anti-Pdx1 (1:10.000, ab47267; Abcam, Cambridge, MA), anti- glucose transporter (Glut)-2 (1:400, Santa Cruz Biotechnology), anti-KGF (1:50, Santa Cruz Biotechnology), anti-phosphoinositide-dependent kinase (PDK)1/2, anti-pan-Akt, and anti-Akt-pThr308. Stainings for PDK1, Akt, and Akt-pThr308 were performed with two different sets of antibodies (Phospho-Akt Sampler Kit, Cell Signaling, ab47267, ab9272, and ab38449 from Abcam). Sections were pretreated with acidic or basic antigen unmasking or trypsination depending on the antibody used. Antibody binding was visualized using a biotinylated secondary antibody and avidin-conjugated peroxidase (ABC method; Vector Laboratories, Burlingame, CA) and 3,3'diaminobenzidine tetrachloride (DAB) as a substrate.

BrdU labeling experiments were performed as described with 3-h pulse 5-bromo-2-deoxyuridine (BrdU) (31). Western blot analyses of KGF (Santa Cruz Biotechnology, 1:500), PDK1/2, PDK-pSer24, Akt, Akt-pThr308, and Akt-pSer473 (Phospho-Akt Sampler Kit, Cell Signaling) expression in whole pancreatic lysates were performed as described with the addition of EGTA (1 mM) and Na3VO4 (1 mM) to the tissue lysis buffer (34). All protein samples were normalized to the total protein content (Bradford reagent; Bio-Rad Laboratories, Hercules, CA), and comparable loading, as well as protein transfer after blotting, was confirmed through Ponceau S staining of the respective membranes.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Microinjection of a 3.5-kb AflIII/PvuI DNA fragment containing the rat elastase promoter (22), the murine KGF cDNA, and part of the human growth hormone gene into F2 DBA2xCl57/Bl6 zygotes resulted in 39 transgenic animals (27.5% of the total number of offspring). Expression of the transgene was detected in six mice by using RT-PCR analysis (30% of the transgenic animals). KGF transgene expression in the transgenic animals was restricted to the acinar cells as confirmed by RNA in situ hybridization. We used a fragment of the spliced hGH mRNA that represents the 5' untranslated region in our construct to specifically detect the transgene expression. Intense hybridization signals in the cytoplasma and nucleus of acinar cells of transgenic animals were obtained with the anti-sense hGH probe (Fig. 1F) but not with the corresponding sense probe (Fig. 1G). No hybridization signal was evident in littermate controls (Fig. 1E) as well as in islets of transgenic mice (data not shown).


Figure 1
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  		Fig. 1. Increased size of islets in close proximity to pancreatic ducts in keratinocyte growth factor (KGF) transgenic mice. Pancreatic tissue sections are stained with hematoxylin and eosin from wild-type mice (A) and 3 independent founders (founder F126, B; founder F110, C; and founder F31, D). The average size of islets is increased in transgenic mice (compare A to B, C, and D). Islets in KGF transgenic mice are frequently found in close contact to intralobular (B) and interlobular ducts (arrowhead in B and D). Original magnification is x50. In situ hybridization with the spliced growth hormone anti-sense probe indicates absent hybridization in littermate control (E) and acinar expression in transgenic animals (founder F126, F). No hybridization is observed with the sense probe (founder F126, G). Scale bar indicates 50 µm.

 
Increased number and size of pancreatic islets in the KGF-expressing F0 generation. KGF-expressing founders (generation F0, Fig. 1, B–D) displayed a pronounced increase in the size and the number of pancreatic islets at the age of 120 days compared with littermate controls (Fig. 1A). In transgenic mice, most of the pancreatic islets were found in close proximity to intra- and interlobular ducts (Fig. 1, B and D). Both types of ducts were dilated and displayed increased branching compared with control animals.

The expansion of the endocrine pancreas was further confirmed by morphometric examination of the six transgene-expressing founders compared with littermate controls (Fig. 2A). The islet area accounted for 1.3 ± 0.2% of the acinar area in control animals. This ratio was increased significantly to 3.6 ± 0.9% in KGF transgenic animals (P < 0.05). In the transgenic founder F126, the relation increased to 7.2%. In addition, morphometric analysis revealed an increased number and size of endocrine islets in the F0 generation.


Figure 2
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  		Fig. 2. Morphometric analysis of the islet area and correlation to transgene expression. A: ratio of the islet area to the acinar area is significantly increased in the founder animals (KGFTG+) F126, F31, F6, and F110 compared with F122, F1, and wild-type (WT) controls. B: mRNA amounts were quantified with real time RT-PCR and expressed as {Delta}Ct values. 18S RNA served as control. Multiple regression analysis revealed a correlation between islet to acinar area and transgene expression (r = 0.8213, P < 0.05).

 
The number of islets was increased to 7.5 x 10–5 ± 0.8 (islet number per arbitrary unit areas of exocrine tissue) in transgenic animals compared with 4.1 x 10–5 ± 0.5 in littermate controls (P < 0.5). Furthermore, transgenic animals showed an average islet size of 495 ± 156 (arbitrary unit areas of islet tissue per number of islets) compared with 316 ± 27 in the control animals. However, this difference was not significant due to the variation in the founder generation.

The variability within the six expressing transgenic animals correlated to the mRNA expression level of the transgene as measured with quantitative RT-PCR. The mRNA level was normalized to the levels of 28S rRNA, resulting in {Delta}Ct values for each sample. As shown in Fig. 2B, there was a close correlation (r = 0.8213, P < 0.05) between the transgene expression and the ratio of islet-to-acinar area. On the basis of this phenotype, permanent transgenic lines were established from the offspring of founder F126 (Fig. 1B, highest expression level upon RT-PCR, line XX) and founder F110 (Fig. 1C, moderate expression level upon RT-PCR, line XVIII). Transgenic mice were crossed to the DBA/2OlaHsd (Harlan) background and analyzed after six to nine crosses.

Increase in β-cells in islets of KGF transgenic mice. Transgenic animals and littermate controls of both lines were investigated after birth at the age of 14, 28, and 45 days and at the late stages mentioned below. Transgenic animals showed normal survival rates and no significant differences in body weight, pancreatic wet weight, and the relation of body weight to pancreatic weight (data not shown). Transgene expression did not result in endocrine or exocrine tumor formation. Morphological differences in terms of an increased relation of endocrine-to-exocrine tissue area were only observed in animals older than 45 days. The phenotype described above with a progressive increase in the endocrine compartment was confirmed in both transgenic lines upon histology (data not shown). As shown in Fig. 3A (overview, transgenic line XX, 180 days) the islets in transgenic animals were mainly composed of insulin-positive β-cells. Furthermore, the comparison with the littermate control (Fig. 3B) illustrates the pronounced effect of the transgene expression. The transgene expression was confirmed by immunohistochemistry (Fig. 3C, line XX) and quantified by Western blot analysis (Fig. 3G). As expected from the RNA data reported above, the expression level was higher in line XX compared with line XVIII. Littermate controls did not express substantial amounts of KGF (Fig. 3, D and G). The KGF-receptor (FGF type 2 receptor) was expressed in islet and ductal cells, both in transgenic animals (Fig. 3E) and littermate controls (Fig. 3F). The expression in the islets colocalized with insulin immunoreactivity upon fluorescence staining (Fig. 3, E and F). We used an antibody for the FGFR2 staining that does not discriminate between the FGFR2IIIb and FGFR2IIIc splice variant. Therefore, expression of these splice variants was further evaluated with RT-PCR. RT-PCR of RNA isolated from microdissected islets and pancreatic ducts amplified the FGFR2IIIb but not the IIIc variant in transgenic mice and littermate controls (Fig. 3H).


Figure 3
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  		Fig. 3. Expansion of the β-cell compartment in KGF transgenic lines. Overview sections labeled with insulin demonstrate the increased number and size of islets in transgenic mice (A, line XX, 180 days) compared with the control (B). KGF expression is evident in the acini of transgenic animals (C) and absent in the control (D). Double labeling with insulin (green) and fibroblast growth factor receptor 2 (FGFR2) (red) show coexpression in the islets (yellow overlay) and expression of FGFR2 in pancreatic ducts (asterisk) in the transgenic (E) and control (F) pancreas. Western blot (G) shows overexpression of KGF in both lines (line XX and XVIII) compared with littermates (WT); +, protein extracts from transfected cells as positive control. RT-PCR (H) of microdissected pancreatic ducts (D) and islets (I) detects expression of FGFR2IIIb but not the FGFR2IIIc splice variant; +, e15.5 embryonic mRNA as positive control; amplification of cyclophilin was used as loading control.

 
The composition of islets in transgenic animals and littermate controls was further analyzed with immunohistochemistry on serial sections (line XX, see below, and line XVIII, data not shown). Figure 4 shows the expression of several markers in comparable sized islets in littermate controls (top) and in the transgenic line XX (bottom, 180 days). Islets in transgenic animals are mainly composed of insulin-positive cells (Fig. 4A), whereas the glucagon (Fig. 4B), somatostatin (not shown), and PP-expressing cells (not shown) are not affected upon immunohistochemistry. Insulin staining detected small groups of positive cells in the exocrine tissue of transgenic mice (Fig. 4A, bottom, inset) with no apparent connection to islets upon serial sections. Immunostaining with chromogranin A confirmed the appearance of endocrine cells in the exocrine compartment of transgenic animals (data not shown). The origin of these cells remains to be defined; however, the close proximity to pancreatic ducts has to be noted. Furthermore, glucagon-positive cells seem to be randomly distributed within the islets and not localized at the outer part of the islets (Fig. 4B). Upon double immunofluorescence staining, we were not able to detect intermediate cell types positive for more than one endocrine hormone (data not shown).


Figure 4
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  		Fig. 4. Expression of hormones and endocrine markers. Sections of littermate control (WT) and transgenic line XX (KGF, 180 days) are stained for insulin (A), glucagon (B), Pdx1 (C), glucose transporter (Glut)-2 (D), amylin (E), Nkx2.2 (F), Islet 1 (G), and albumin (H). Insets in C and D: nuclear staining of Pdx1 and membranous staining of Glut-2 in higher magnification. Insulin, Pdx1, and amylin-positive cells are evident in the exocrine compartment of the transgenic mice (KGF; A, C, and E, insets). Likewise, Nkx2.2 and Islet 1-positive cells are evident in the exocrine compartment (KGF; F and G, arrow). H depicts absent albumin-positive epithelial cells intransgenic mice and controls. Original magnification is x50. Relative quantification of mRNA levels for insulin, Pdx1, and glucagon (I) indicates increased levels of insulin mRNA in transgenic animals. Pdx1 and glucagon mRNA level are comparable with exception of a single high Pdx1 mRNA expressing animal.

 
Further characterization was aimed to evaluate the expression of factors involved in proper β-cell function. In this respect, expression of Pdx1 and the glucose transporter Glut-2 are known to be essential for glucose-dependent insulin secretion. Nuclear expression of Pdx1 is evident in transgenic animals and controls. Furthermore, Pdx1 staining detected small groups of positive cells in the exocrine tissue of transgenic mice (Fig. 4C, bottom, inset). Glut-2 staining demonstrates membranous immunoreactivity in transgenic animals and controls (Fig. 4D).

Insulin staining indicated the presence of endocrine cells in the exocrine compartment. This was further confirmed by immunostaining for amylin (Fig. 4E). Amylin-positive cells are evident in the exocrine compartment of transgenic animals (Fig. 4E, inset) but not in littermate controls. Along these lines, we found expression of transcription factors in single cells between the acinar cells such as Nkx2.2 (Fig. 4F, arrow head), islet 1 (Fig. 4G, arrow head), and Pax6 (data not shown). Hepatic differentiation of pancreatic cells was described in transgenic mice overexpressing KGF under control of the rat insulin promoter. Immunostaining with anti-albumin antibodies (Fig. 4H) shows albumin immunoreactivity associated with blood vessels. In our model, we did not find evidence of hepatic differentiation based on this staining.

Quantitative RT-PCR analysis of whole tissue lysate revealed an increased expression of insulin mRNA in pancreatic samples of both transgenic lines compared with littermate controls (Fig. 4J). The expression of glucagon is comparable in transgenic animals and littermate controls, indicating that the number of {alpha}-cells is unchanged. Interestingly, the mRNA expression of Pdx1 was not elevated in transgenic animals, despite the overall increase in the number and size of the islets. This comparable level of Pdx1 and Glut-2 expression was further confirmed in Western blot analysis (data not shown).

Induction of the PKB/Akt pathway in islets of transgenic animals. In the following set of experiments, we evaluated Ki67 staining and BrdU incorporation in transgenic animals (line XX, see below and line XVIII, data not shown) and littermate controls. The number of Ki67-positive cells was slightly increased in both transgenic lines (Fig. 5A, line XX and data not shown, age 90 days) compared with the controls (Fig. 5B). Ki67 immunostaining indicated proliferating cells within the islets and the pancreatic ducts of transgenic mice. This increase was found both at early stages (day 45) as well as in aged animals (>180 days). BrdU incorporation (Fig. 5C, line XX; D, wild-type, 3-h BrdU pulse, 90 days) confirmed Ki67 staining. Using a 3-h BrdU pulse, we did not detect a significant number of BrdU-positive cells in littermate controls, whereas transgenic mice showed approximately one to three BrdU-labeled cells per islet.


Figure 5
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  		Fig. 5. Proliferation and activation of protein kinase B (PKB)/Akt in KGF transgenic mice. Ki67 staining (A, B) and 5-bromo-2-deoxyuridine (BrdU) labeling (C, D, 3-h pulse) indicate the presence of proliferating cells in the islets and ducts in transgenic mice (arrowheads; A and C, line XX, 90 days) compared with controls (B and D). Western blot (E) indicates upregulation of phosphorylated Akt pThr308 in line XX and comparable expression levels of phosphoinositide-dependent kinase (PDK)1, PDK pSer24, pan-Akt, and Akt pSer473 in transgenic mice and in controls. In situ, expression of PDK1 (F and G) and pan-Akt (H and I) is comparable in transgenic animals (G, I, and K, line XX, 340 days) and in controls (F, H, and J). K depicts increased Akt pThr308-positive cells (arrowhead) in the islets of transgenic animal compared with the control (J). Original magnification is x50.

 
The lack of a profound increase in proliferation and the previously reported phenotype of PKB/Akt transgenic mice (30) prompted us to evaluate the activation of the Akt pathway in the islets of transgenic animals. Figure 5E shows comparable expression and activation (in terms of Ser24 phosphorylation) of PDK1 upon Western blot analysis in KGF transgenic animals and controls. Likewise, the expression of Akt and phosphorylation of Akt at Ser473 is not affected. However, Western blot analysis revealed increased phosphorylation of Akt at Thr308 in the pancreas of transgenic animals, indicating activation of the Akt pathway in these mice. (Fig. 5G). Akt activation was further analyzed with immunohistochemistry. PDK1 (Fig. 5, F and G) and pan-Akt (Fig. 5, H and I) showed a comparable expression pattern in the endocrine and exocrine compartment in the transgenic mice (Fig. 5, G and I) and in controls (Fig. 5, F and H). Interestingly, expression of both proteins was more pronounced in the endocrine compartment. Figure 5K demonstrates the number of Akt-pThr308-positive cells in islet cells in the pancreas (line XX, 340 days) compared with the nearly absent staining in littermate controls (Fig. 5J).

Disturbed glucose tolerance and insulin secretion in KGF transgenic mice. We further evaluated fasting blood glucose levels in transgenic animals and littermate controls older than 200 days to analyze metabolic effects of the described phenotype (Fig. 6A). Littermate controls had blood glucose levels below 100 mg/dl after 12 h of fasting even in animals older than 500 days. In contrast, elevated blood glucose levels were found in transgenic animals from both lines (Fig. 6A). In particular, animals older than 1 year were diabetic with glucose levels up to 200 mg/dl. In a more detailed analysis, we evaluated blood glucose in younger animals (120 to 280 days) both during a 24-h fasting period as well as after intraperitoneal injection of dextrose. No hypoglycemia was observed during the fasting period (Fig. 6B). Furthermore, these animals did not show hyperglycemia under regular diet at the beginning of the fasten period (Fig. 6B, first data point) or under the fasting condition. This excluded that the animals were apparently diabetic at this stage. Blood glucose levels increased in both transgenic lines compared with the controls after challenging the animals with dextrose. Whereas blood glucose returned to normal values within 120 min in the controls, transgenic animals had elevated blood glucose levels at this time point. This finding was more pronounced in line XX. Insulin serum levels were measured in a subset of these animals during glucose challenge. Littermate controls responded with an increased serum insulin level to elevated blood glucose levels (Fig. 7). The serum insulin in the transgenic animals was comparable to the littermate controls in the animals with normal blood glucose (Fig. 7). However, transgenic animals did not respond to the elevated blood glucose with increased insulin release to the blood stream as indicated by the trend line included in Fig. 7. Due to the low number of animals, we did not perform statistical analysis of this effect. However, these findings, together with the pathological response to the glucose challenge, strongly suggest impairment of glucose-stimulated insulin secretion in both KGF transgenic lines.


Figure 6
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  		Fig. 6. Blood glucose levels in transgenic animals. A: fasting blood glucose increases with age in both transgenic lines (line XX, {blacktriangleup}, and line XVIII, {blacksquare}, compared with littermate controls depicted with bullet; blood glucose measured after 12-h fast). During a 24-h fasting period no obvious difference is observed in blood glucose (animals 120 to 280 days, line XX, {blacktriangleup}, and line XVIII, {blacksquare}, compared with littermate controls depicted with bullet). B: glucose challenge (2 mg/g body wt ip) results in increased blood glucose levels in both lines. Blood glucose levels remain elevated in transgenic animals 120 min after glucose challenge (line XX, {blacktriangleup}, and line XVIII, {blacksquare}, compared with littermate controls depicted with bullet).

 

Figure 7
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  		Fig. 7. Serum insulin compared with serum glucose in KGF transgenic animals. Serum insulin and glucose measured during the glucose challenge shown in Fig. 6. Transgenic lines XX and XVIII are depicted with {blacktriangleup} and {blacksquare} and compared with littermate controls depicted with bullet. Trend line indicates the glucose-dependent increase in serum insulin in littermate controls (WT) and the missing insulin response in transgenic animals (KGF).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Ectopic expression of KGF induces an expansion of the endocrine compartment in the pancreas of transgenic mice. Although the expression of the transgene was directed to the acinar cells, particularly, the duct cells and endocrine tissue exhibit dramatic morphological changes, indicating that KGF is efficiently secreted and acts in a paracrine fashion. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue increased compared with littermate controls. Frequently, we found endocrine islets in close proximity to pancreatic ducts. Immunohistochemical staining of the pancreas of KGF transgenic mice indicates that the enlarged islets are composed mainly of insulin-positive cells, suggesting a selective expansion of the β-cell compartment. We did not observe obvious differences of {alpha}-, {delta}-, or PP cells in terms of hormone expression upon light microscopy and immunohistochemistry. Furthermore, overexpression of KGF in our model does not result in intermediate cell types expressing multiple endocrine hormones as described for IFN-{gamma} transgenic mice (12). Interestingly, the phenotype of transgenic overexpression of KGF observed in this study differs from recent findings in several aspects. Overexpression of KGF directed to pancreatic islets resulted in proliferation of pancreatic duct cells and the emergence of hepatocytes within the islets of Langerhans. The effect on the β-cell lineage was rather limited (15). We did not find evidence of hepatic differentiation on the basis of the morphology and albumin staining in our transgenic mice. Delivering KGF using a proinsulin promoter is different to the ectopic expression of the transgene under the control of the elastase promoter. Furthermore, different experimental settings and the genetic background might account for the observed differences.

Differentiation of endocrine cells from pancreatic duct cell or a putative stem cell in this compartment has been hypothesized in several publications (4, 10, 26, 35). The regeneration of the endocrine compartment (4, 35) and the model of IFN-{gamma} transgenic mice (13) serve as paradigms for this hypothesis. Interestingly, KGF was identified as growth factor induced under these experimental conditions (1, 15). This observation led to the generation of the proinsulin promoter-driven KGF transgenic mice discussed above (15). Furthermore, the influence of the pancreatic mesenchyme in general and the KGF/FGF receptor type 2, in particular, on embryonic differentiation is well recognized (6, 7, 11, 18). In this respect, KGF is able to mimic the effect of embryonic mesenchyme on pancreatic epithelium in terms of progenitor cell proliferation and suppression of exocrine differentiation (7). Our results strongly suggest that mesenchymal-derived growth factors such as KGF are important for the maintenance and probable expansion of β-cells in the adult mouse. Along these lines, we demonstrate cells positive for insulin and endocrine-specific transcription factors such as Pdx1, Nkx2.2, islet 1, and Pax6 outside the islet compartment. Furthermore, KGF induces proliferation of pancreatic duct cells in vivo. KGF and FGFR2 are overexpressed in human pancreatic cancer (14). This indicates that the KGF/FGF2 receptor signaling pathway might contribute to pancreatic tumorigenesis in vivo. However, overexpression of KGF alone was not sufficient to induce pancreatic tumors in our model.

The concept that β-cells differentiate from duct cells (or a putative precursor) under physiological conditions was questioned more recently (5). In these experiments, Melton and colleagues (5) provided clear evidence that β-cells arise from self-duplication, at least under the experimental condition applied in their study. Further evidence for the concept of β-cell self-renewal comes from several in vivo models focusing on the role of the PI3 kinase/PKB/Akt pathway on β-cell maintenance (2, 21, 29, 30), suggesting that replication of differentiated β-cells is the major mechanism for β-cell regeneration in the adult pancreas. Along these lines we observed a slight but consistent increase in β-cell proliferation and increased PKB/Akt phosphorylation at threonine 308 in pancreatic islets of KGF transgenic mice. This most likely attributes to a direct activation of PKB/Akt through the KGF/FGF type 2 receptor signaling pathway as described, e.g., in alveolar type II cells in vivo (24, 25, 27). It should be noted that the islet area was increased approximately threefold, whereas proliferation as measured by BrdU incorporation and Ki67 labeling was only slightly increased in KGF transgenic animals. The discrepancy might be explained by increased survival of insulin-positive cells as reported for Akt transgenic animals (2). However, given the evanescent nature of apoptosis in vivo, we were not able to assess the possible contribution of decreased apoptosis to the increased cell number observed in KGF transgenic mice.

RIP-Cre-driven inactivation of PTEN resulted in a moderate, approximately twofold increase in β-cell number with no apparent alteration in β-cell function, e.g., with an intact glucose-sensing capacity in this expanded β-cell population (21, 29). Furthermore, this genetic manipulation renders animals resistant to β-cell injury. Therefore activation of the PKB/Akt pathway represents an attractive therapeutic target in diseases with loss of β-cells. Activation of the PBK/Akt pathway through a soluble growth factor such as KGF represents a potential therapeutic strategy. Overexpression of KGF does not appear to change the qualitative normal course of cellular differentiation based on the expression of endocrine-specific markers such as insulin, glucagon, somatostatin, PP cell, and β-cell-specific markers such as islet 1, Nkx2.2, Pdx1, and Glut-2. However, our functional analysis revealed functional defects in glucose homeostasis rather than metabolic improvement in KGF transgenic mice. Along the same lines, overexpression of the active Akt1 mutant in transgenic mice resulted in a decreased maximal secretory capacity per unit β-cell mass as indicated in a more detailed analysis of the expanded pancreatic islet (30). We observed an expansion of the β-cell compartment before the onset of disturbed glucose tolerance. Therefore, it is unlikely that primary effect of KGF is a downregulation of insulin secretion and hyperglycemia and that the increase of the β-cell compartment is only a result of the hyperglycemia. The more likely explanation for the disturbed glucose tolerance and insulin response to blood glucose in our model would be quantitative differences in the expression of proteins essential for β-cell function such as Pdx1 and Glut-2, as well as the altered distribution of {alpha}-cells within the islet. Comprehensive functional analysis is therefore wanted before a particular pathway is taken in consideration as therapeutic target.


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This work was supported, in part, by grants from the Bundesministerium für Bildung und Forschung, IZKF, C4 (to R. Schmid) and from the Deutsche Forschungsgemeinschaft, SFB518/A10 (to M. Wagner).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Wagner, Dept. of Gastroenterology and Endocrinology, Univ. Ulm, Robert Koch Str. 8 89081 Ulm, Germany (e-mail: martin.wagner{at}uni-ulm.de); R. Schmid, Dept. Internal Medicine II, TU Munich Ismaninger Str. 22, 81675 Munich, Germany (e-mail: roland.schmid{at}lrz.tum.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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