Posters: Islet Biology-Beta Cell Growth and Differentiation

1576-P

Collectrin, a Transmembrane Glycoprotein Expressed in [beta]- Cells, Is a Target of Hnf1 [alpha] and Is Developmentally Regulated in the Embryonic Pancreas

PINAR AKPINAR, MARKUS STOFFEL. New York, NY

Hnf1 [alpha] is a homeodomain transcription factor that is necessary for proper islet development and function. Hnf1[alpha]- deficient animals, an experimental mouse model of MODY3, are characterized by impaired insulin secretion and reduced islet mass. We compared gene expression in islets from Hnf1 [alpha] null mice and wildtype littermate control animals. Expression of collectrin, a transmembrane glycoprotein and homologue of ACE2, was reduced 16- fold in Hnf-1[alpha] deficient mice compared to controls. Collectrin is selectively expressed in the kidney and the [beta]-cells of the pancreatic islets. We identified three putative Hnf1 binding sites in the promoter of the collectrin gene, which are conserved in rat, human and mouse. Electrophoretic mobility shift assay (EMSA) revealed that Hnf1[alpha] can bind to these sites. Hnf1[alpha] is a strong activator of the collectrin promoter in vitro and mutations in the Hnf1 binding sites abolish transcriptional activation. Immunohistochemical analysis of different stages in the developmental regulation of collectrin indicates that it colocalizes with insulin positive [beta]-cells in E18.5 pancreas and in adult islets. In contrast, in earlier developmental stages of days E15.5 and E13.5 collectrin is coexpressed in glucagon positive cells but does not colocalize with insulin. These findings suggest that collectrin is a target of HNF1[alpha] and may contribute to the defect in insulin secretion in MODY3. Furthermore, the developmental expression pattern suggests a role for collectrin in endocrine pancreas development.

1577-P

Streptozotocin-Based Selection of Insulin-Producing BTC-tet Cells with Improved Defense Properties

KONSTANTIN BLOCH, MARINA VOROBEYCHIK, KARINA YAVRIANS, PNINA VARDI. Tel Aviv, Israel

Beta-cell lines constitute a potential source of genetically engineered insulin-producing cells to replace difficult-to-obtain human tissue for pancreatic islet transplantation. Recently, our group have reported the selection strategies for engineering insulin- producing RIN cells with improved resistance against diabetogenic toxins (streptozotocin -STZ, alloxan) and hydrogen peroxide. The main obstacle of RIN cells is that these insulinoma cells are poorly differentiated. In the present study we describe selection procedure for obtaining highly differentiated BTC-tet cells with multiple resistance to different toxins. Mouse BTC-tet cells are both significantly more differentiated and more sensitive to diabctogenic toxins compared with RIN insulinoma cells. The selected BTC-tet/D cells with improved defense properties were obtained by repeated exposure of parental BTC-tet cells to increasing concentration of STZ (1 mM and 2 mM). The 2.5-fold increased resistance of BTC-tet/D cells to 2 mM STZ compared with parental cells was confirmed by two different cytotoxicity assays with MTT and Alamar blue. In addition, the selected cells displayed multiple dose-dependent resistance to the cytotoxic effect of alloxan, hydrogen peroxide and nitric oxide. The enhanced defense properties of the selected cells were not associated with any significant changes in insulin content. However, selected cells displayed reduced basal insulin release and enhanced insulin response to a 16.7 mM glucose in the presence of 1.0 mmol/l isobutylmethylxanthine compared with parental BTC-tet. Another effect of the STZ-based selection procedure was associated with increased proliferation activity of BTC-tet/D cells. Microscopic examination revealed a flattened morphology of selected cells compared with a spherical shape of parental BTC-tet cells. In conclusion, repeated exposure to STZ induces selection of highly maturated BTC-tet cells with multiple resistance to diabetogenic toxins, hydrogen peroxide and nitric oxide.

1578-P

Islet Neogenesis Therapy (INT) Reverses Diabetes in Psammomys obesus

THORAB. BODVARSDOTTIR, CARSTEN F. GOTFREDSEN, TONY CRUZ, ALEKSANDRA PASTRAK, ANNETTE M. PERREGAARD, JACOB S. PETERSEN. Bagsvaerd, Denmark; Toronto, ON, Canada

Loss of islet cells is a hallmark of late stage type 2 diabetes. Both EGF and Gastrin have been implicated in islet growth/ neogenesis. We therefore investigated if co-administration of an EGF (E1)- and a Gastrin (G1) analouge, referred to as Islet Neogenesis Therapy (INT), could be used to treat severely type 2 diabetic animals, as Psammomys obesus, a desert gerbil that spontaneously develops diabetes when fed a high energy (HE) diet.

Fifteen diabetic (morning blood glucose (BG) 17.6 0.9 and 19.8 1.8mM for vehicle and INT, respectively) Psammomys obesus that had been fed HE diet for 4 weeks where allocated to 2 groups with the same mean HbA^sub 1c^. Vehicle (VE) or INT (G110g/kg/day + E1 3g/kg/ day distributed on 2 daily doses) were administered i.p for 14 days and the study was terminated 10 days after the last treatment. BG and body-weight (BW) was measured once daily and HbA^sub 1c^ once weekly. At the end of the study, beta-cell mass was measured immunohistochemically.

All animals (N=8) in the VE group remained diabetic throughout the 24 day study period (morning BG at end of study was 20.6 1.2mM). Five out of 7 animals in the INT group became normoglycaemic during the treatment with a morning BG at end of study of 3.9 0.4mM. Two of the INT treated animals remained hyperglycaemic throughout the study. The 2 animals in the INT group that did not respond to treatment had been diabetic from the beginning of HE feeding. After the end of the study, HbA^sub 1c^ was increased by 28% (from 8.6 0.3 to 11.0 0.2) in the VE treated whilst it was reduced by 5% (from 8.5 0.5 to 8.1 0.9, including the 2 non- responders) in the INT treated animals. BW was not different between the groups.

Immunohistochemical analysis of beta-cell mass revealed that INT treated had approx. 2 fold higher beta-cell mass as well as higher levels of insulin in the beta-cells, as compared to VE treated Psammomys obesus.

We conclude that INT has the potential to become a treatment that can revert the disease process or slow the progression of diabetes.

1579-P

Developmental and Physiological Roles of the Notch2/RBP-J in the Pancreas

JUNJI FUJIKURA, TOSHIKI SAITO, KIMINORI HOSODA, HIROSHI IWAKURA, TSUTOMU TOMITA, MICHIO NOGUCHI, HIROAKI MASUZAKI, TATSUYA HAYASHI, SHIGERU CHIBA, YOSHIO HAMADA, TASUKU HONJO, KAZUWANAKAO. Kyoto, Japan; Tokyo, Japan; Aichi, Japan

Notch signaling is involved in regulating the balance between cell differentiation and stem cell proliferation during the development of numerous tissues. It has been shown that Notch signaling could alter cell fate decisions in pancreatic epithelium, mainly based on the experiments of transgenic mice expressing constitutive active Notch1 or Notch3 gene driven by Pdx1 promoter. Because the generalized knock-out mouse models for Notch1, Notch2 or RBP-J resulted in early embryonic lethality, the relative importance of Notch signaling in the regulation of pancreas development remains to be unknown. To determine the roles of Notch signaling, we analyzed the Notch2 expression patterns in pancreas by X-gal staining of Notch2-lacZ knock-in mice and then addressed the role of Notch2 or RBP-J in pancreas development and glucose homeostasis by inactivating each gene specifically in the pancreas. In the day 15.5 mouse embryo, Notch2 was expressed in the ductal cells, believed to be the source of pancreatic stem cells. In the adult, strong expression was detected in the cells of ducts and blood vessels, and weak expression was also detected in the islet cells. Notch2 or RBP- J floxed mice were bred with Pdx.Cre. transgenic mice harboring the Cre-recombinase transgene driven by the Pdx1 promoter. We observed almost complete deletion of the floxed Notch2 or RBP-J gene exons. Phenotypes of these pancreas specific conditional kock-out mice are currently under investigation.

1580-P

Pancreatic Islet-Specific Expression of an IGF-I Transgene Increases Islet Cell Mass in GHR^sup -/-^ Mice

YUBIN GUO, YARONG LU, KAREN COSCHIGANO, JOHN KOPCHICK, ZHENGYITANG, KATIEROBERTSON, JUN-LI LIU. Montreal, QC, Canada; Athens, OH

Both GH and IGF-I promote pancreatic islet cell growth, inhibit apoptosis and are potentially involved in normal islet growth and maintenance. We and others have recently demonstrated that GH receptor gene deficiency (GHR^sup -/-^) causes diminished pancreatic islet cell mass and insulin production and elevated insulin sensitivity. As IGF-I gene expression is nearly abolished in GHR^sup -/-^ mice, we sought to determine whether these effects were due to the severe deficiency in IGF-I. In order to correct the deficiency, we have generated transgenic mice that express the IGF-I gene under the direction of the rat insulin promoter I (RIP-IGF) and studied whether it can rescue islet defects in GHR^sup -/-^ mice. Studies using Northern blot and immunohistochemistry revealed that the IGF- I transgene is specifically expressed in pancreatic islets of the RIP-IGF mice (which are GHR^sup +/+^). These animals exhibit normal growth and development and possess no abnormalities in glucose homeostasis and insulin p\roduction, except that the average size of their pancreatic islets was enlarged 1.9 fold in female RIP-IGF-I vs. wild-type littermates. As reported, GHR^sup -/-^ mice exhibit diminished islet cell mass; in particular the ratio of islet cell mass/body weight was decreased 50% vs. wild-type littermates. They also exhibited increased insulin sensitivity but impaired glucose tolerance. In double transgenic GHR^sup -/-^ mice that express the IGF-I transgene (GHR^sub -/-^+RIP), islet cell mass was increased 3.8 fold (1.05 0.27 vs. 0.27 0.08 mg, n=10, P=0.01), glucose tolerance was significantly improved, yet the levels of serum insulin, pancreatic insulin mRNA and insulin sensitivity were unchanged vs. GHR^sup -/-^ alone. Interestingly, the double transgenic mice demonstrated a partial rescue in growth vs. GHR^sup – /-^ mice, at 2-3 months of age (i.e. -12% increased body weight, P<0.05). Thus, local expression of an IGF-I transgene restored pancreatic islet cell mass in GHR^sup -/-^ mice, supporting the notion that IGF-I mediates the GH-induced growth promoting effect on pancreatic islet cells.

1581-P

Inducers of Islet Neogenesis from Adult Human Pancreatic Progenitors

STEPHEN C. HANLEY, AL-MALEEK JAMAL, MARK A. LIPSETT, LAWRENCE ROSENBERG. Montreal, QC, Canada

We have previously reported the capacity of cultured islets to transform into duct epithelial cysts (DECs) containing putative islet progenitors. The extent of their neogenic potential has not been fully elucidated. We sought to examine the effects of previously established neogenic agents in this model. Human islets, isolated according to the Edmonton protocol, were embedded in type 1 collagen and cultured in DMEM/F12 with 10% FBS, 10 ng/mL EGF, 24 mU/ mL insulin and 100 ng/mL cholera toxin (CT). By day 10, islets having differentiated into DECs were characterized via microarray, real-time PCR and immunohistochemistry as lacking endocrine hormones, while expressing duct markers (cytokeratins, carbonic anhydrase, CFTR) and the proposed progenitor cell markers acid [beta]-galactosidase and neurogenin-3. Further culture of DECs in the above medium without CT, but with a range of GLP-1 or exendin-4 concentrations, showed no dithizone-positive budding. This is consistent with microarray analysis suggesting GLP-1 receptors are underexpressed in DECs. Combined treatment with 50 nM gastrin + 10 ng/mL HGF produced dithizone-positive structures in 20.27.3% of DECs (p<0.05 vs control). Treatment of DECs with 167 nM islet neogenesis- associated protein^sup 104-118^ (INGAP) induced budding from 39.29.5% of DECs (p<0.01 ). A dose-response study was conducted in an analogous canine model: 16.7, 167 and 1670 nM INGAP produced budding from 20.35.5%, 28.44.1% and 29.65.2% of DECs (p<.05 vs control). DECs treated with 167 nM INGAP and 2, 4 or 8 L/mL [alpha]- INGAP antibody had dithizone-positive budding reduced by 19.73.3%, 58.10.4% and 78.22.0% relative to INGAP alone (p<0.01), confirming INGAP's neogenic role. As well, treatment of DECs with wortmannin abrogated INGAP's neogenic effects, therefore implicating PI3'K. Finally, culture with gastrin + HGF + INGAP induced budding in 38.62.5% of human DECs (p<0.01 vs control), demonstrating that gastrin + HGF and INGAP are not synergistic, perhaps due to common signaling. These data suggest that several means exist to induce in vitro islet neogenesis from these differentiated islet progenitors.

1582-P

Role of Pax4 during Adult Beta Cell Regeneration

FRIEDRICH HARDER, RAINER MUSSMANN, CORD DOHRMANN, MATTHIAS AUSTEN. Goeftingen, Germany

A common feature in diabetes (type I, LADA, late stage type II) is a deficit in functional beta cells. As an alternative appoach to the transplantation of islets or in vitro differentiated stem cells we are focusing on the identification of factors which can stimulate beta cell neogenesis in vivo.

An important focus of our work is the Pax4 gene. Pax4 has been shown previously to be required for beta cell development during mouse embryogenesis. We also show that it enhances the differentiation of mouse embryonic stem cells towards a phenotype similar to beta cells in vitro and improves function after transplantation in vivo. However, as for other factors controlling embryonic pancreas development comparably less is known about Pax4 function during beta cell and/or islet regeneration in adults. Since ductal cells appear to be important in human beta cell regeneration we have analyzed the endocrine differentiation from ductal epithelium and the role of Pax4 in this process.

We have analyzed Pax4 expression in pancreatic cells of mice under normal and regenerating conditions. We found that Pax4 is activated in ductal or duct-associated cells which express genes indicative of endocrine differentiation. In order to further characterize the role of Pax4 we have used overexpression in mouse ductal cells. Pax4-overexpression in these cells resulted in a significant upregulation of endocrine differentiation markers under appropriate conditions.

We conclude that Pax4 represents a useful marker of beta cell regeneration and can enhance endocrine differentiation of ductal epithelial cells.

1583-P

Human Pancreatic Islet Hormone-Expressing Cells May Not Be Terminally Differentiated

ANANDWARDHAN A. HARDIKAR, CHIJU WEI, BERNICE MARCUS-SAMUELS, ELIZABETH GERAS-RAAKA, BRUCE M. RAAKA, MARVIN C. GERSHENGORN. Bethesda, MD

We demonstrated that insulin^sup -^/cytokeratin-7^sup +^,-19^sup +^ human PANC-1 cells differentiate to hormone-expressing islet- like cell aggregates (ICAs) when exposed to a defined serum-free media (SFM). The insulin^sup +^ cells within ICAs are cytokeratin- 7^sup -^,-19^sup -^ and show very low proliferation by BrdU/Ki-67 staining and cell cycle analysis. When EGF (100 ng/ml) was added to ICAs in SFM, cells began to proliferate and show cytokeratin -7^sup +^, -19^sup +^ cells, suggesting that proliferation is accompanied by de-differentiation. When these cells are again exposed to SFM, they form hormone-expressing ICAs. This process of differentiation, de-differentiation and re-differentiation can be carried out for at least 3 rounds of de-differentiation.

As PANC-1 cells are cloned from a pancreatic carcinoma, we used postnatal human islets and cultured them in serum containing CMRL medium to determine if an insulin-producing cell can de- differentiate and re-differentiate. Cells grow out from islets and at ~14 days in vitro (passage 0), represent an insulin^sup -^/ C- peptide^sup -^ and vimentin^sup +^ fibroblast-like population of human islet-derived precursor cells (hIPCs), which still express insulin mRNA (0.1% of fresh islets) by quantitative real-time RT- PCR (qRT-PCR). hIPCs proliferate for at least 28 doublings and can be induced to differentiate to ICAs that express insulin mRNA and C- peptide. To investigate the origin of hIPCs, we studied islets during the initial 14 days in culture. By combined in situ hybridization and immunocytochemistry we were able to detect cells in transition that co-express insulin transcript and vimentin protein, though most of the cells in the parental islets were vimentin^sup -^ but co-expressed C-peptide and insulin transcript. Further analysis of the population with BrdU staining, immunocytochemistry and single-cell qRT-PCR adds to the notion that insulin-producing cells de-differentiate to hIPCs. Our data provides evidence that [beta]-cells may de-differentiate to a precursor cell population, which can expand and re-differentiate into hormone expressing ICAs.

1584-P

Limiting Artefact: The Use of Gluco-Conditioning To Create an In Vitro Primary Islet Model

MOIRA J. HARRISON, ADRIAN J. BONE. Brighton, E. Sussex, United Kingdom

In vitro experiments have limited application to the understanding and modelling of diabetes since cell lines are phenotypically very different from primary pancreatic islets. Cells are routinely maintained in media containing supra physiological concentrations of glucose, typically 10-25mM/l. Human islets have limited availability and animal use is being discouraged. Previously we have used clusters of MIN6 cells (pseudoislets) as a model for primary islets. Pseudoislets (PIs) phenotypically resemble primary islets, maintain their islet-like morphology and respond to external stimuli over extended culture (28+ days). Cell-lines and pseudoislets are normally maintained in 25mM glucose-containing media. The drawbacks to studying chronic high glucose exposure effects in cells habituated to high glucose are obvious. We took the PIs through selective passage and serum withdrawal to condition them to physiological levels of glucose. Cells were serum-deprived for 3 days to synchronise cell cycle and passaged over 4 weeks in decreasing glucose concentrations (22-5.5mM glucose). Cells were frozen at -70 C (21 days) then cultured in media containing 10%FCS and 5.5mM glucose. Cells and PIs were cultured for 7 days in the presence of 5.5, 11 or 22mM glucose. Apoptosis, viability and necrosis were determined using a combination of Hoescht/Propidium Iodide dyes. Pl viability was similar at 5.5mM and 11mM glucose levels but significantly reduced at 22mM glucose (73 4% v 62 3%, p < or =0.05). Cells compared to PIs were not significantly different at 5.5mM glucose. At 11 mM glucose, viability remained the same but cell apoptosis was significantly lower compared to PIs (9 1% v 17 2, p< or = 0.05). At 22mM glucose levels cell viability was greater than PIs (78 3% v 62 3, p< or = 0.01) and lower apoptosis (17 2% v 28 3, p< or =0.05. Necrosis levels were not significantly different at 5.5mM, 11mM or 22mM glucose. Gluco-conditioning enabled culture of MIN6 cells in physiological levels of glucose which was further enhanced when they were configured as pseudoislets leading to the establishment of a superior in vitro model of primary pancreatic islets.

1585-P

Exenatide Does Not Cause Pancreatic Islet CellProliferative Lesions in Rats and Mice Following 2-year Exposure

RICHARD HILES, THOMAS CARPENTER, DAVID SEROTA, KENNETH SCHAFER, PAUL ROSS, DENNIS NELSON, MARLON REBELATTO. San Diego, CA; Mattawan, MI; Greenfield, IN

Exenatide (synthetic exendin-4) and GLP-1 have been reported to increase [beta]-cell mass in laboratory animals via stimulation of [beta]-cell neogenesis and proliferation. The concern is that uncontrolled [beta]-cell proliferation could occur with chronic exposure to any agent that stimulates GLP-1 receptors resulting in neoplastic lesions. In these 2-year carcinogenicity studies (following ICH guidelines) of exenatide in rats and mice (65 male, 65 female/group; Sprague-Dawley rats and CD-1 mice), animals received daily subcutaneous injections of placebo (vehicle, 2 groups) or exenatide (18, 70 and 250 g/kg/day). Exenatide doses were equivalent to approx. 6, 25 and 90 times the systemic exposure expected from a 20 g/day human dose. Pancreatic tissue samples from all animals were evaluated microscopically by board certified veterinaiy pathologists and subsequently peer reviewed.

1586-P

Mechanisms Underlying [beta] Cell Mass Expansion in Young Pre- Obese Zucker Rats

THOMAS L. JETTON, MINA PESHAVARIA, JAMES A. LAUSIER, KYLA LAROCK, BROOKE L. LARMIE, JACK L. LEAHY. Burlington, VT

The mechanisms that underlie [beta] cell expansion in response to insulin resistance are largely unknown. Using the well-studied Zucker fatty (ZF) rat that exhibits a striking increase in islet mass compared to their lean littermates, we have investigated the age-related [beta] cell mass dynamics pre- and post- the weaning period and the onset of insulin resistance. Although ZF pups were not born with heightened [beta] cell mass, by 3 wks of age their values were 1.4-fold increased. This timepoint was several days before differences in body weight and plasma insulin levels could be detected. Five wk-old ZF rats exhibited a > 3.5-fold increase in [beta] cell mass, which is several weeks before the reported onset of insulin resistance. Unexpectedly, no differences were observed in islet [beta]cell replication rates between the ZF and control rats at any age. Instead [beta] cell neogenesis was markedly increased from 3-wks of age through adulthood. This was indicated by increases in ductal nuclear PDX-1 staining and significantly heightened numbers of small [beta] cell clusters. To gain insight into the molecular mechanisms underlying islet neogenesis in young ZF rats, we investigated key insulin signaling molecules IRS-2 and Akt. IRS- 2 and activated (phospho-) Akt levels in the ductal epithelium were dramatically elevated in 3.5-wk old ZF rats. Another important finding was that PDX-1+ cells showed activated Akt immunostaining. Surprisingly, activated Akt immunoreactivity was also increased in islet [beta] cells of ZF rats. BAD is a proapoptotic factor and an established substrate of Akt, and is inhibited upon phosphorylation. Accordingly, increased phospho-BAD staining was observed in [beta] cells of ZF rats. Thus, our findings suggest that Akt signaling has important but distinct cell-specific roles during early mass augmentation in the young Zucker rat: maturation of [beta] cell precursors during ductal neogenesis, and enhanced survival of islet [beta] cells. ADA Funded Research

1587-P

Enhanced Ductal Insulin Signaling in Association with Increased [beta] Cell Neogenesis in Glucose-Infused Rats

THOMAS L. JETTON, JAMES L. LAUSIER, MINA PESHAVARIA, AFSHIN SALSALI, JACK L. LEAHY. Burlington, VT

The molecular basis of [beta] cell growth and regeneration is not resolved. Recent studies suggest that the insulin-signaling pathway may mediate compensatory [beta] cell expansion upon increased insulin requirements. Key intermediates in this cascade, IRS-2 and Akt, have been implicated with roles in the growth, survival, and neogenesis of [beta] cells. In vivo glucose infusions is a well known model of [beta] cell mass expansion. Accordingly we studied pancreatic insulin signaling in rats following a 2 day infusion of 20% glucose which resulted in a mild increase in plasma glucose at 24 h that totally normalized by 48 h. Remarkably, [beta] cell mass was increased 3-fold in these rats. We next investigated the mechanisms that contributed to this rapid growth response. Islet [beta] cell proliferation decreased during this period, and [beta] cell size increased modestly by 20%. In contrast, islet neogenesis was augmented in these rats. Despite the lack of increased ductal proliferation, the ducts exhibited increased nuclear PDX-1 expression, as well as enhanced numbers of insulin+ and PYY+ cells. Furthermore, a >2.5-fold increase in the prevalence of small [beta] cell clusters were observed. We then examined the role of the insulin signaling cascade in duct neogenesis. Ducts from glucose- infused rats showed increased IRS-2 and phosphotyrosine immunoreactivity as well as a marked enhancement of activated (phosphorylated) Akt and [beta]-catenin levels. Importantly, phospho- Akt consistently colocalized with intensely-labeled PDX-I+ and Pax6+, insulin-negative cells. Our results suggest that the rapid [beta] cell mass increase in these rats is not the result of [beta] cell proliferation, but instead, modest [beta] cell hypertrophy. Enhanced duct neogenesis appears to provide a major contribution of new [beta] cells. Our data also suggest that Akt signaling in the duct epithelium initiates early [beta] cell differentiation from precursors during [beta] cell mass adaptation following a glucose infusion. ADA Funded Research

1588-P

P57KIP2 Expression in Rat Pancreas during Development

SAMEERA, KASSEM, ESTHER P. HABER, ILANA ARIEL, TAMARA POTIKHA, BENJAMIN GLASER. Jerusalem, Israel

P57KIP2 (CDKN1C) belongs to the CIP/KIP family of cyclin dependent kinase inhibitors. It is an important inhibitor of several G1 cyclin/Cdk complexes, causing cell cycle arrest. Loss or underexpression of p57KIP2 has been related to several malignancies and to Beckwith-Wiedeman Syndrome. We previously reported that p57KIP2 was selectively expressed in human beta cells and that loss of expression in the focal form of hyperinsulinism of infancy is associated with increased beta-cell proliferation. In the current study we examined p57KIP2expression during different stages of rat development. Wistar rat embryos at gestational days E17.5-E21.5 and rats at ages 1 to 9 weeks old were examined. Using immunohistochemistry, we evaluated major rat organs for p57kip2 immunostaining. Double staining with insulin was performed on sections containing pancreatic tissue.

During embryonic life, p57KIP2 was highly expressed only in stromal cells of the pancreas. This expression decreased after birth. At one week of age, weak immunostaining was also seen in the exocrine tissue, whereas at 2 weeks of age and thereafter, high levels of expression were demonstrated exclusively in the exocrine pancreas. Insulin positive cells did not stain for p57KIP2 at any stage during rat development. In skeletal muscle, heart, kidney, and lung, p57KIP2 expression was high in embryonic life and decreased after birth. In the liver, p57KIP2 staining was demonstrated only at the days E19.5 and E21.5. In testis, cells that stained positive for p57KIP2 were: interstitial cells during embryonic life, spermatogonia at 1 week and spermatides at 7 weeks old.

A distinct spatial and temporal expression of p57KIP2 was demonstrated in different tissues, including the pancreas during rat development. In contrast to our findings in human, no expression of p57KIP2 was detected in beta-cells during any stage of rat development. These findings suggest that p57KIP2 does not play an important role in beta-cell development in the rat. This is in direct contrast to our previous findings that this protein may play a pivotal role in controlling proliferation of the human beta-cell.

1589-P

Unique Expression Pattern of Pdx-1 and Ngn3 during the Regeneration of Pancreatic [beta] Cells after Streptozotocin Treatment

SHOKO KODAMA, TATSUYA KONDO, TETSUSHI TOYONAGA, SHOEN KUME, EIICHI ARAKI. Kumamoto, Japan

It has been reported that progenitor cells exist in the adult pancreas and convert to endocrine cells under pathological conditions. Pdx-1 (pancreatic and duodenal homeobox-1) and Ngn3 (neurogenin3) arc important transcription factors which are involved in pancreatic development and [beta] cell differentiation. During the regeneration of adult [beta] cells, Pdx-1 and Ngn3 may also play an important role. The aim of this study is to investigate the expression of Pdx-1 and Ngn3 during [beta] cell regeneration. We employed transgenic mice which express GFP under the control of Pdx- 1 promoter, investigated the expression of Pdx-1 and Ngn3 mRNA and observed the localization of Pdx-1-positive cells by immunohistological examination during [beta] cell neogenesis after STZ treatment. Pdx-1 mRNA level examined by real-time PCR was increased to 2 fold of basal level at 48 and 72h after STZ injection, and was decreased to the basal level at day 4, then it was again increased significantly to 6 fold at day 5 to 7. The Ngn3 mRNA was not detected until 24h after STZ treatment, and was slightly expressed at 48h, and then expressed obviously at 72h. Immunohistological analysis revealed that almost all of the [beta] cells were destroyed and a small number of Pdx-1-positive cells were detected in pancreatic ducts after 24h of STZ treatment. Pdx-1- positive cells were also observed in extra-ductal area at 48 and 72h, and Pdx-1-positive islet-like clusters appeared with the decrease of Pdx-1 expression in ducts at day 7. Exendin-4 injection increased Pdx-1 expression to 1.5 fold of that of uninjected group at 48 and 72h after STZ treatment. In conclusion, during the [beta] cell regeneration which occurs after STZ treatment, progenitor cells that existed in duct or residual islets expressed Pdx-1 biphasically an\d Ngn3 transiently, and the regeneration efficiency could be increased by exendin-4 injection. This knowledge will help us to understand [beta] cell regeneration and to invent new therapeutic approach toward diabetes.

1590-P

Reduced [beta]-Cell Area Associated with Decreased Cyclin D and CDK4 Expression in Irs1/Irs2 Double Knockout E 18.5 Embryos

NORIE KOWATARI, KAZUYUKI TOBE, RYO SUZUKI, KENTARO SAKAMOTO, HIROTAKA WATADA, KAZUHIRO ETO, NAOTO KUBOTA, YASUO TERAUCHI, RYUZO KAWAMORI, TAKASHI KADOWAKI. Tokyo, Japan

[beta]-cell mass has been reported to be decreased in the pancreas of patients with type 2 diabetes mellitus. We previously demonstrated that while Irs1^sup -/-^ mice showed growth retardation and normal glucose tolerance associated with [beta]-cell hyperplasia to compensate for insulin resistance in skeletal muscle, Irs2^sup -/ -^ mice developed diabetes associated with [beta]-cell growth failure and insulin resistance in the liver and obesity. In order to clarify the role of Irs1 and Irs2 in islet [beta]-cell development, we intercrossed Irs1^sup +/-^Irs2^sup +/-^ double-heterozygous mice (on a C57BL/6J x CBA hybrid background) to obtain offspring of various genotypes. At 18.5 days post coitum (d.p.c.), embryos of all genotypes including Irs1^sup -/-^Irs2^sup -/-^ showed viability with expected frequencies. The body weights of the embryos depended on the number of wild-type IRS alleles (Irs1^sup +/+^Irs2^sup +/+^: 1.22 0.03 g, Irs1^sup +/+^Irs2^sup -/-^: 1.00 0.02 g, Irs1^sup -/- ^Irs2^sup +/+^: 1.01 0.02 g, Irs1^sup -/-^Irs2^sup -/-^: 0.54 0.01 g; means SE, at least 8 animals per genotype). We then examined the islet cell morphology in these embryos. Histological analysis revealed that the [beta]-cell mass and islet organization were preserved in the Irs1^sup -/-^Irs2^sup +/+^ and Irs1^sup +/ +^Irs2^sup -/-^ embryos. However, Irs1^sup -/-^Irs2^sup -/-^ double- homozygous embryos showed disrupted islet architecture with insulin positive cells scattered around glucagon positive cells. In addition, the mean size of insulin positive cells was significantly reduced in these Irs1^sup -/-^Irs2^sup -/-^ embryos. Interestingly, immunostaining analysis revealed that the expression of cyclin D and CDK4 was more severely reduced in the Irs1^sup -/-^Irs2^sup -/-^ embryo [beta]-cells. Our results suggest the important role of IRS signaling pathway on the regulation of [beta]-cell size and mass, and islet development at the embryonic stage.

1591-P

Committing Murine Embryonic Stem Cells to Early Endocrine Pancreas In Vitro

H. TERESA KU, NAN ZHANG, ATSUSHI KUBO, RYAN O’CONNOR, MINWEI MAO, GORDON KELLER, JONATHAN BROMBERG. New York, NY

Recent efforts have been directed at the generation of insulin producing cells using the embryonic stem (ES) cell-embryoid body (EB) culture system, to provide islet cells for transplantation and cure of type I diabetics. The extent of endoderm pancreas differentiation was not addressed nor specified in previous reports. There are two non-allelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominatly insulin II, small amounts of insulin I, and no glucagon. In our studies, a panel of genetic markers was used to assess the in vitro commitment of murine ES cells toward the endoderm-derived pancreas, and to distinguish insulin-expressing cells of this lineage from other lineages. We found that ES-derived EBs cultured in the presence of stage specific concentrations of monothioglycerol and 15% FCS, followed by serum-free conditions, gave rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker) and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin peptides, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin/C-peptide expressing cells co-express glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of nicotinamide and exendin-4 in culture augmented insulin I expression by 88-fold, using quantitative real time RT-PCR analysis. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.

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WITHDRAWN

1593-P

Pituitary Tumor Transforming Gene-Null Mice Exhibit Early Compensatory [beta]-Cell Neogenesis and Later Inverted Pancreatic Islet [beta]/[alpha]-Cell Ratio Coinciding with Diabetes Onset

SERGIO LI CALZI, SHLOMO MELMED. Los Angeles, CA

[beta]-cell mass is dynamic and maintained during development and after diabetogenic insults through regeneration of pre-existing [beta]-cells and/or neogenesis by ductal cell trans- differentiation. Pituitary Tumor Transforming Gene (pttg), a mammalian homolog of yeast securin, regulates sister chromatid segregation in mitosis. Previously we showed that pttg-/- male mice display pancreatic [beta]-cell hypoplasia and insulin deficiency leading to adult-onset diabetes, implying that compensatory mechanisms maintain young KO mice euglycemic.

pttg-/- islets were smaller (121892232 vs 255514274 m^sup 2^; p<0.05) than WT. Pancreatic sections from 3-week pttg-/- mice expressed significant immunoreactivity for insulin in cells lining 2 of 14 pancreatic ducts identified and PDX-1 in 1 of 14. Thus, diabetes is delayed in pttg-/- due to compensatory islet neogenesis. This phenomenon was not observed in older pttg-/- mice or in WT at any age. Diabetes occurs at ~8 months in males (~85% penetrance) and ~15 months in females (>50%). Sections derived from 3-week pttg-/- males displayed a [beta]/[alpha]-cell ratio similar to WT (pttg-/- [beta]/[alpha]:64/36 vs WT:67/33). Diabetic 8-month pttg-/- males showed an inverted [beta]/[alpha]-cell ratio (pttg-/[beta]/ [alpha]:28/72 vs WT:79/21). Pre-diabetic 8-month pttg-/- females showed a [beta]/[alpha]-cell ratio similar to WT (pttg-/- [beta]/ [alpha]:59/41 vs WT:69/31). Diabetic 15-month pttg-/- females had an inverted [beta]/[alpha]-cell ratio (pttg-/- [beta]/[alpha]:18/82 vs WT:78/22). Control pre-diabetic 15-month pttg-/- females [beta]/ [alpha]-cell ratio was similar to WT (pttg-/- [beta]/[alpha]:54/ 46).

Young pttg-/- male mice are a useful genetic model exhibiting compensatory [beta]-cell neogenesis which could underlie development of the late hypo-insulinemic phenotype, as a consequence of age- related equilibrium disruption, caused by rising demand for insulin in response to body mass increase. The temporal onset of pttg-/- diabetes is directly linked to [beta]/[alpha]-cell pancreatic islet ratio reversal. The [beta]-cell appears to be preferentially hypoplastic in the absence of securin, indicating selective cell- cycle disruption leading to insulin-deficiency and diabetes.

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Reduced [beta]-Cell Replication Decreases [beta]-Cell Mass in Placental Insufficiency-Intrauterine Growth Restricted (PI-IUGR) Fetuses

SEAN W. LIMESAND, JAN JENSEN, WILLIAM W. HAY, JR.. Aurora, CO; Denver, CO

Human fetuses with intrauterine growth restriction have less pancreatic endocrine tissue and exhibit decreased hormone secretion in response to glucose. This pathology may impose limitations to insulin secretion in later life, as infants of low birth weight have an increased incidence of non-insulin dependent diabetes mellitus. Placental insufficiency is a primary complication contributing to IUGR infants by restricting nutrient supplies to the fetus and secondarily the growth and development of the pancreas. Therefore, we studied an ovine model of placental insufficiency and IUGR to determine factors contributing to decreased [beta]-cell mass in control and PI-IUGR fetuses by examining [beta]-cell replication and apoptosis. Pregnant ewes were exposed to elevated ambient temperatures (40C for 12h:35C for 12h) for 80 days of gestation (from 24 to 82% of gestation) to create PI-IUGR fetuses. The fetuses were autopsied at ~135 days or 92% of gestation. Fetal weights were reduced by 51%, paralleled by a 57% reduction in pancreas weights: 1.70.2g in PI-IUGR fetuses and 4.00.3g in control fetuses. The percent insulin area was 5.30.6% in control fetuses (n=6), but only 2.60.1% (P<0.01) in PI-IUGR fetuses (n=8). These two components decreased [beta]-cell mass 78% in the PI-IUGR pancreases (45.05.9 mg vs 204.325.5 mg). Apoptosis in [beta]-cells, determined by TUNEL immunohistochemistry, was unchanged by nutrient deprivation, 1.90.4% in PI-IUGR compared to 2.00.5% in controls, [beta]-cell proliferation was determined by PCNA immunostaining (S-phase); there was no difference (P=0.1) between PI-IUGR (13.51.4%) and control (10.20.8%) fetuses. However, [beta]-cells positive for phosphorylated histone H3, a marker for mitosis, showed a 72% decrease in PI-IUGR fetuses (0.150.02% vs 0.540.04%). Therefore, [beta]-cell replication is decreased in PI-IUGR fetuses, likely by nutritional regulation of the cell cycle that decreases [beta]-cell mass. This research was supported by NIH F32DK60300-1 and NIH RO1HD60773.

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Evidence for Regulation of [beta]-Cell Mass Expansion in Response to Induction of Islet Neogenesis

MARKLIPSETT, MAURO CASTELLARIN, JULIE LEMAY, LAWRENCE ROSENBERG. Montreal, QC, Canada

Recently much effort has been focused on the restoration of a glucose-responsive, insulin secreting [beta]-cell mass for individuals suffering from diabetes mellitus. One such innovative approach is the neogenic expansion of endogenous [beta]-cell mass. A critically important question is whether the long-term expansion of [beta]-cell mass is subject to homeostatic control mechanisms. Support for this notion comes from our studies of partial duct obstruction of adult hamster pancreata in which [beta]-cell mass expansion is mediated by the upregulation of Islet Neogenesis Associated Protein (INGAP). Using average islet size (m^sup 2^) and islet density (#/mm^sup 2^) as surrogate\s of [beta]-cell mass, there is an initial rapid expansion of the endocrine tissue (average rate of change of insulin-stained area/mm^sup 2^) of 12.7%/day from day 0 to 7. The average rate of expansion then decreases with time (5.3%/day from day 7 to 42; and 2.8%/day from day 42 to 56). This apparent plateau of [beta]-cell mass expansion is also seen following administration of the biologically active INGAP pentadecapeptide^sup 104-118^ (500 g/day) to euglycemic hamsters. From 0 to 10 days of treatment, the average rate of [beta]-cell mass expansion is 148 g/day, whereas, from day 10 to 30 the average rate of expansion decreases to 45 g/day. When INGAP peptide is administered to normoglycemic CD-1 mice the [beta]-cell mass initially expands (day 34: 1.840.36 vs 0.830.09 mg, p<0.05), but returns to control levels at 90 days, despite the presence of persistent islet neogenesis as indicated by a 4-fold increase in duct-associated focal areas of neogenesis (8.52.1 vs 2.00, p<0.05) and a 2-fold increase in duct-associated islets (33.87.6 vs 15.83.3, p<0.05). These data suggest that in the presence of ongoing islet neogenesis, homeostatic regulatory mechanisms intervene to match [beta]-cell mass to the prevailing metabolic requirements.

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Persistent Upregulation of PDX-1 in Pancreatic Progenitors Inhibits Exocrine Differentiation and Increases Insulin-Producing Cells

TAKESHI MIYATSUKA, YOSHIO FUJITANI, HIDEAKI KANETO, TOSHIHIKO SHIRAIWA, KEN KATO, TAKA-AKI MATSUOKA, YOSHIHISA NAKATANI, NAOTO KATAKAMI, DAN KAWAMORI, SHIN-ICHI GOROGAWA, MUNEHIDE MATSUHISA, YOSHIMITSU YAMASAKI, MASATSUGU HORI. Suita, Osaka, Japan

It has been reported that PDX-1-expressing cells give rise to all three types of pancreatic tissue: endocrine, exocrine, and duct. In exocrine and ductal cells, PDX-1 gene expression is downregulatcd after late embryonic development. We have previously generated transgenic CAG-CAT-PDX1 mice, which constitutively express PDX-1 gene under the control of chicken beta-actin gene (CAG) promoter after the removal of the floxed stuffer sequence (CAT) by Cre- mediated recombination. To determine whether sustained expression of PDX-1 could change the cell fates of pancreatic precursors, we crossed the CAG-CAT-PDX1 mice with Ptf1a-Cre mice, which express the Cre recombinase driven by the Ptf1a (PTF1-p48) gene promoter in precursors of all three pancreatic cell types. In the Ptf1a-Cre/CAG- CAT-PDX1 mice, PDX-1 was expressed in over 80% cells in pancreatic acini as well as islets at postnatal day 10. The pancreas of the transgenic mouse was markedly small accompanied by a significant decrease of acinar cells. Immunohistochemical analysis revealed much more insulin-positive cells outside the islets than control littermates. These data suggest that downregulation of PDX-1 in Ptf1a-expressing pancreatic progenitor cells is crucial for exocrine differentiation and that constitutive upregulation of PDX-1 in the cells promotes endocrine differentiation.

1597-P

Engineering Hematopoeitic Cells To Produce and Secrete Insulin

AMBER L. MOSLEY, MEGAN L. SAMPLEY, JOHN P. WILLIAMS, SABIRE OZCAN. Lexington, KY

Defects in insulin production result in hyperglycemia, which is the major cause for the secondary complications of type II diabetes. An attractive strategy to treat diabetes associated with a lack of or decreased insulin production is to engineer non-beta cells to produce and secrete insulin in a glucose concentration-dependent manner. Recent data indicate that bone marrow cells can give rise to islet beta-like cells, suggesting the possibility that bone marrow cells can be engineered to serve as surrogate beta cells. Furthermore, previous data indicate that monocyte/macrophage precursor cells in bone marrow are glucose sensitive and express the glucose transporter GLUT-2 and glucokinase.

We found that macrophage precursors of bone marrow osteoclasts are glucose sensitive and express and secrete insulin when infected with an adenovirus containing the beta-cell specific transcription factor Pdx-1. Infection of RAW264.7 cells, which are macrophage precursors of osteoclasts with an adenovirus expressing the transcription factor Pdx-1 led to production of insulin in these cells compared to control cells infected only with GFP containing virus. Moreover, R AW264.7 macrophage cells expressing Pdx-1 also secreted insulin in a glucose-dependent manner. These data suggest that bone marrow contains a subpopulation of cells that are glucose- sensitive and have the secretory machinery to secrete protein in a glucose-dependent manner. We are currently testing whether lentiviral expression of human insulin in bone marrow can be used as a therapeutic treatment for overtly diabetic NOD mice.

1598-P

Roles for GPCRs in the Differentiation of Human Pancreatic Precursor Cells

CHRISTOPHER M. MULLA, CHIJU WEI, YORAM ORON, BERNICE MARCUS- SAMUELS, ELIZABETH GERAS-RAAKA, BRUCE M. RAAKA, MARVIN C. GERSHENGORN. Bethesda, MD

Cell surface receptors play an important role in the differentiation of pancreatic pre-curser cells into mature hormone producing cells. We have established a culture system of human islet- derived precursor cells (hIPCs) from cadaveric pancreata. hIPCs can form insulin-producing, islet-like cell aggregates (ICAs) when monodispersed with trypsin and cultured without serum. Similarly, pancreatic cancer PANC-1 cells also form ICAs. Protease-activated receptors (PARs) have four members, but only PAR-2 expression has been reported previously in the pancreas. RNA transcripts for both PAR-1 and PAR-2 are present in human adult islets, fetal pancreas, PANC-1 cells and hIPCs, but human islets express more PAR-2 than PAR- 1, while hIPCs express more PAR-1 than PAR-2. Activation of PAR-1 by thrombin or PAR-2 by its peptide agonist leads to inositol phosphate (IP) second messenger generation and raises intracellular free calcium in both hIPCs and PANC-1 cells. Importantly, stimulation of PAR-1 accelerates ICA formation. Lastly, we found that trypsin stimulates IP generation and intracellular free calcium, suggesting trypsin might be critical for the formation of ICAs. Since trypsin is expressed in the exocrine pancreas, our data suggest that stimulation of PARs by trypsin might play a role in islet formation during embryonic development. Thyrotropin-releasing hormone (TRH) knockout mice experience mild hypoglycemia as well as hypothyroidism suggesting that TRH may also play an important role in pancreatic development (Hashida et. al 2002). Using quantitative real time RT- PCR, we have detected transcript levels for TRH and its receptor (TRHR) in human fetal pancreas that would be consistent with a role for TRHR signaling during pancreatic development. In contrast, we have detected TRH but not TRHR transcripts in ICAs from PANC-1 cells and hIPCs and in adult islets. We believe that TRHR signaling may also play an important role in fetal islet development and are continuing efforts to assess its possible role in adult islets.

1599-P

The Expression of Neurogenin3 and Nkx6.1 Can Differentiate AR42J- B13 Cells into Insulin Producing Cells

TAKESHI OGIHARA, HIROTAKA WATADA, REI KANNO, TOYOYOSHI UCHIDA, NOSEKI IWASHITA, YASUSHI TANAKA, MICHAEL S. GERMAN, ITARU KOJIMA, RYUZO KAWAMORI. Tokyo, Japan; San Francisco, CA; Maebashi, Japan

Neurogenin3 (Ngn3) is an essential and sufficient transcription factor for endocrine cell differentiation in pancreas. Ngn3, by itself can drive pancreatic epithelia cells into [alpha] cells, not [beta] cells, implying that there are some factors necessary for [beta] cell differentiation in the transcriptional cascade downstream of Ngn3. To explore these factors, using AR42J-B13 cells as a model of the differentiation of pancreatic islet cells, we investigated the change of the expression of transcription factors. The results of the RT-PCR analysis demonstrated that the expression of glucagon, but not insulin was induced by forced expression of Ngn3 using adenovirus system in AR42J-B13 cells, like in pancreatic epithelial cells. Pancreatic progenitor cell marker, Ptf1a was disappeared by the expression of Ngn3. Pdx1 gene expression was once decreased, but recovered to basal level later. The expression of pancreatic endocrine transcription factors such as Pax4, Pax6, Nkx2.2 and NeuroD were remarkably increased. These changes of gene expression pattern by the forced expression of Ngn3 seemed to be reasonable considering the process of the differentiation in pancreas. During this differentiation process, we were not able to detect expression of a pancreatic [beta] cell factor, Nkx6.1 in any time. Therefore, next, we tried to induce the expression of Nkx6.1 in AR42J-B13 cells by adenovirus system. The forced expression of Ngn3 and Nkx6.1 induced the expression of insulin, not glucagon in AR42J-B13 cells. This expression of insulin was confirmed by RT-PCR analysis and RIA using cell extracts from AR42J-B13 cells. However, the forced expression of LacZ or Nkx6.1 without Ngn3 did not induce the expression of insulin nor glucagon. Confirming that Ngn3 promotes the differentiation into endocrine cells, our findings demonstrate that Nkx6.1 is sufficient to drive the cell fate to [beta] cell after the beginning of the differentiation process induced by Ngn3 in AR42J-B13 cells.

1600-P

p8 Is Associated with Glucose-Induced Pancreatic Beta-Cell Growth and Enhances Proliferation of Adult hMSC-TERT Stem Cells

GUNTER PATH, ANNE OPEL, JOCHEN SEUFERT. Wuerzburg, Germany

Aim: Transplantation of pancreatic beta-cells can restore insulin production in type 1 diabetics but is limited by a lack of donor organs. Generation of beta-cells from human pancreatic islet- derived nestin-positive stem cells (N+SC) represents a novel strategy to support availability of transplant material. Since growth of cultured primary N+SC is limited, we are testing the proliferation associated protein p8 as a candidate for exp\ansion of these cells in vitro.

Methods: p8 mRNA and protein expression was investigated with RT- PCR and immunohistochemistry (IHC). p8 expression in INS-1 beta- cells during 25 mM glucose-induced growth was quantified by Western blot analysis (WBA) after 3, 24, 48, 72, 96, 120, and 144 hrs in culture. In parallel, cell numbers were assessed at time points indicated. IHC and WBA were performed using a peptide-derived polyclonal monospecific rabbit p8 antiserum. Growth of human bone marrow stem cells (hMSC-TERT) transfected with a CMV promoter driven p8-IRES-GFP or IRES-GFP (mock) vector was measured by FACS counting of GFP-positive cells.

Results: p8 is expressed in all principle pancreatic differentiated cell types and N+SC. Glucose-induced p8 expression in INS-1 beta-cells increased rapidly to a maximum after 48 h and then decreased continuously to a stable level. Cell numbers displayed an identical but delayed time course (maximum after 96 h). In addition, overexpression of p8 in hMSC-TERT cells resulted in significantly increased cell growth as compared to mock transfected controls.

Conlusion: These data indicate 1) p8 is a glucose-induced mediator of pancreatic beta-cell growth without oncogenic properties in vivo and 2) overexpression with p8 is well suited to enhance growth of adult stem cells in vitro. In combination with our previously presented nestin-specific vectors, p8 may be a tool to expand human pancreatic islets-derived N+SC. The activity of these vectors will be silenced by differentiation-induced phenotype switch from N+SC to nestin-negative beta-cells, thereby avoiding uncontrolled cell growth.

1601-P

Growth-Factor Enhanced Transdifferentiation of Acinar Cells to Pancreatic [beta]-Like Cells in Short-Term Culture

MINA PESHAVARIA, AIDA HABIBOVIC, JAMES A. LAUSIER, BROOKE L. LARMIE, JACK L. LEAHY, THOMAS L. JETTON. Burlington, VT

Although [beta]-cell neogenesis in the adult is thought to originate mainly from duct epithelial precursors, recent studies suggest that the acinar compartment may also serve as a source of new [beta]-cells.

Using a previously described in vitro rat culture system for acinoductal transdifferentiation, we now demonstrate an enhanced transdifferentiation (TD) potential from acinar tissue to [beta]- like cells in the presence of the growth factors, HGF, bFGF and EGF (HFE). Normally discarded acinar tissue after rat islet isolation, consisting largely of acinar cells (>99%) but also single and/or small [beta]-cell clusters (>0.5%) grew as spheroids that progressively lost their acinar characteristics in the presence of serum within 24h. However, in the presence of HFE, the culture became enriched in hybrid amylase+/Insulin+(Ins) and Ins+ cells within 24h. Hybrid cells failed to co-express ductal/endocrine markers suggesting there was no intervening duct-like intermediate step. Although HGF is a proven mitogen for pancreatic [beta]-cells, proliferation of [beta]-cells in the presence of HFE was not detected through 72h of culture period suggesting that TD occurred without replication. These [beta]-like cells exhibited increased expression of [beta]-cell transcription factors, PDX-1 and MafA, however, their localization pattern was cytoplasmic, suggesting partial maturation. Cytoplasmic expression of p48 was also maintained despite decreased levels of amylase protein and enzyme activity. Interestingly, a further increase in the number and maturation of Ins+ cells was observed in islet co-culture experiments with HFE-treated spheroids where expression of PDX-1 and MafA was nuclear after 48h. These results suggest that the acinar tissue may represent another source for [beta]-cell neogenesis and also that additional islet-borne growth factors may play a role in [beta]-cell differentiation from acinar cells.

1602-P

The Proliferation Associated Protein p8 Interacts with a Novel Splice Variant of the DEAD Box RNAHelicase DDX18 in Pancreatic Beta- Cells

ANDREAS PETER, ANNE OPEL, MATHIAS D. BRENDEL, REINHARD G. BRETZEL, JOCHEN SEUFERT. Wuerzburg, Germany; Giessen, Germany

Background/Aims: Previously we have shown that the nuclear protein p8 is expressed in insulin producing pancreatic beta-cells. Glucose regulated expression of p8 is associated with cell proliferation. p8 is also expressed in endocrine pancreatic progenitor cells. To further understand the molecular mechanisms by which p8 promotes pancreatic beta-cell proliferation, the aim of this study was to identify proteins physically interacting with p8 in human pancreatic islet cells. Methods/Results: Using a GAL 4- based yeast-two-hybrid system to screen a human pancreas cDNA library we detected a splice variant of the c-myc regulated DEAD box polypeptide 18 (DDX18) as an interaction partner of p8. We confirmed physical interaction between human p8 and DDX18 performing co- immunoprecipitation experiments. The interaction partner of p8 was characterized as a novel splice variant of DDX18 expressed in human pancreatic islets using RT-PCR and sequencing analysis. DEAD box proteins are putative RNA helicases. Specifically DDX18 has been implicated in the regulation of c-myc-dependent cell proliferation. The DDX18-gene is a direct target of c-myc and its expression is upgegulated upon mitogenic stimulation. In Drosophila loss of its homologue protein pitchoune, causes a small size phenotype, resembling the phenotypes of c-myc mutants. This identifies DDX18 as a protein mediating c-myc-dependent growth control. Conclusions: We have identified a novel splice variant of the c-myc regulated DEAD box polypeptide DDX18 as a direct interaction partner of the proliferation associated protein p8 in human pancreatic islets. These results provide a molecular link between p8 and c-myc induced proliferation pathways in insulin producing cells. We propose, that neogenesis of pancreatic beta-cells is in part mediated by the protein p8 through activation of the c-myc regulated protein DDX18.

1603-P

Rapid Reorganization of Extracellular Matrix andTGF-[beta] Expression Is Important in the Process of Pancreas Regeneration after Partial Pancreatectomy

MARIE RHEE, KUN HO YOON, SUN HEE SUH, SUNG YOON JUN, HYE SOO KIM, JE HO HAN, SOON JIB YOO, KI HO SONG, JONG MIN LEE, BONG YUN CHA, KWANG WOO LEE, HO YOUNG SON, SUNG KOO KANG. Republic of Korea

To understand the roles of transforming growth factor (TGF)- [beta] & its related signals on pancreas regeneration process in partial pancreatectomized rat pancreas over time. We performed 90% partial pancreatectomy (Px) in 100 g SD rats & pancreata were removed 0, 1, 2, 3, 5, 7 & 14 days after Px. Sections were stained with TGF-[beta] antibody. RT-PCR for TGF-[beta], connective tissue growth factor (CTGF), [alpha]-SMA, fibronectin, angiotensin converting enzyme (ACE), angiotensin II receptor (ATIIR) TIMP 1, TIMP2, MMP-2, & MMP-9 were performed. TGF-[beta] was weakly expressed in islets & duct epithelial cells in normal pancreas and after Px, remarkably increased in common pancreatic duct & duct epithelial cells in the foci of regeneration on day 3, then gradually disappeared. TGF-[beta] mRNA expression reached a peak value on day 2, decreased from day 5 & finally returned to the basal level at day 14. CTGF, [alpha]-SMA, fibronectin, & ATIIR mRNA expressions were similar to TGF-[beta] expression pattern. ACE mRNA reached to peak from day 1. TIMP 1 mRNA reached to the peak from day 1 & gradually decreased to control level by day 14, whereas the expression of TIMP 2 mRNA was maximal on day 5. MMP-2 mRNA reached a peak value on day 2, whereas MMP-9 mRNA was elevated on day 1, & gradually decreased. In conclusion, after Px, ACE, TIMP 1 & MMP-9 expressions were reached to peak from day 1. Then, TGF-[beta], CTGF, [alpha]-SMA, fibronectin, ATIIR, TIMP 2 & MMP-2 expressions were followed & reached to peak at day 3. All the signals started to be decreased from day 5 & finally returned to the basal levels at 14 days after Px. Taken together, rapid reorganization of extracellular matrix & TGF-[beta] expression is important for duct cell proliferation at the early stages of pancreas regeneration.

1604-P

Insulin-Positive Cells in Pax6 Knockout Mice: Do They Function as [beta]-Cells?

ALDO ROZZO, SHI-BING YANG, TIZIANA MENEGHEL-ROZZO, MARJAN RUPNIK. Goettingen, Germany

A [beta]-cell is an insuline-positive cell that releases this hormone in response to glucose and tolbutamide stimulation. It is characterized by a large resting K^sub ATP^ conductance and by an almost complete inactivation of voltage-activated Na+ current at resting membrane potential.

The Pax6 transcription factor is important for CNS, eye and endocrine pancreas development and Pax6 knockout (KO) mice die at birth. It is a strong candidate for specifying the lineage of [alpha]-cells and its lack results in the almost complete absence of [alpha]-cells and misshaping of the islets of Langerhans. The aim of this work was the functional characterization of the [beta]-cells in this almost pure insulin-positive cell clusters.

We established a new preparation to study the function of [beta]- cells in late embryonic pancreas by slicing the en-block organ to 100 m slices. In the wild type (WT) littermate the cultured pancreatic islets survive many weeks and at least during the first two weeks they maintain the excitability and releasing competence comparable to the acute slices. The exocrine tissue instead disappears after few days in culture.

The confocal analysis of the Pax6 KO pancreas confirmed the absence of proper islets. Not only the endocrine cells were dispersed clusters instead of dense islets, in addition the ganglia were missing and the innervation was apparently aborted showing fibers without a target. Using patch-clamp we showed that neither the electrophysiological properties nor the excitability of these insulin-positive cells were similar to WT [beta]-cells. In particular the half inactivation of the Na+ current wasnot in the physiological range (WT -90 mV vs KO -45 mV); the resting membrane potential was more positive (WT -65 mV vs KO 50 mV) and finally the K^sub ATP^ channel conductance was significantly lower (WT 3-5 nS vs KO 0.1 nS; intracellular ATP clamped to 0.5 mM). We conclude that insulin containing cells in Pax6 KO mice are not functional [beta]- cells missing the usual channels/pumps pool that give them the ability to respond to glucose.

1605-P

Role of Hyperinsulinemia in the Regulation of Islet [beta]-Cell Regeneration and Differentiation in a New Mouse Model

SUPARNA A. SARKAR, JOHN C. HUTTON. Denver, CO

We have developed an animal model using sustained hyperinsulinemia/hypoglycemia that leads to reversible involution of the endocrine pancreas to study changes in gene expression and transcriptional studies to provide insight into the intracellular events that regulate [beta]-cell mass. Black Swiss and NOR females mice between ages 6-8 weeks were surgically implanted with Alzet osmotic pumps subcutaneously. The pumps loaded with 50-500 IU of Humulin-R dispensed insulin at a fixed-rate of 0.25 ml/hr and were explanted after 14 days to terminate the treatment. Control mice received similar pumps loaded with diluant only. Dose-duration study indicated that 0.3-0.45 IU Humulin/day caused significant hypoglycemia without jeopardizing animal morbidity and mortality. Impaired glucose tolerance test was noted during insulin exposure. Glycemic control gradually returned to normal over a period of several days. Histochemical analyses revealed vacuolated islets and decreased insulin staining in the [beta]-cells of mice exposed to high dose of insulin. Morphometric analyses by point-counting method showed increase in [beta]-cell mass 1-day after the termination of insulin treatment. Concurrently, small increase in glucagon producing [alpha]-cell mass was also noted. Cell mass changes returned to normal 6-days after the termination of treatment. Further analysis revealed that increase in [beta]-cell mass could be partially accounted for by the increase in mean [beta]-cell area or hypertrophy that was found to be significantly different (p=0.03) from the control group. Increase in mean cell volume was also noted (p=0.04) in the treated group. Our physiological data is consistent with the previously published insulinoma-like tumor implantation and resection rodent models. Morphometric analyses indicated that increase in [beta]-cell mass could be partially explained by increase in mean [beta]-cell cross-sectional area and volume (hypertrophy). Relative roles of islet cell replication, islet neogenesis and apoptosis in pancreatic regeneration in this model is being studied in greater detail.

1606-P

A BAC Transgenic Mouse Model of Neurogenin3 Expression

YOSHINORI SHIMAJIRI, DAVID SCHEEL, SHUHONG ZHAO, YASUHIRO KOSAKA, MICHAEL S. GERMAN. San Francisco, CA

The expression of Neurogenin3, a basic helix-loop-helix transcription factor, determines which cells will become islet cells during pancreatic development. To study how Neurogenin3 expression is regulated, we developed a novel transgenic mouse line in which surrogate markers can be used to gauge Neurogenin3 expression in vivo. Starting with a 164 kb bacterial artificial chromosome (BAC) containing the human gene encoding Neurogenin3 (NEUROG3), we replaced the coding sequence for Neurogenin3 with a bicistronic gene composed from two readily detectable marker genes, secreted alkaline phosphatase (SEAP) and enhanced green florescent protein (EGFP), separated by a viral internal ribosome entry site (IRES). As an initial trial, we tested the bicistronic transgene in vitro by transient transfection in the mouse pancreatic ductal cell line, mPAC. 1 kb of 5′ flanking DNA from the NEUROG3 gene was sufficient to generate an EGFP signal in transfected cells and measurable SEAP activity in the media. Inclusion of 3′ flanking sequences from the NEUROG3 gene strongly enhanced SEAP expression, suggesting that the NEUROG3 gene may contain multiple, dispersed transcriptional enhancers, and underlining the importance of using the large NEUROG3 BAC for producing transgenic mice. Three independent transgenic mouse lines were produced with the NEUROG3-SEAP/GFP BAC. Transgenic embryos were assessed for transgene expression at embryonic days E14.5 and E16.5. Cells expressing EGFP could be detected in the pancreas at or near the ducts. SEAP secretion rates were assayed by culturing isolated pancreatic buds on extra-cellular matrix substitute (Matrigel) from E12.5 embryos. SEAP activity in the bud culture media increased linearly over time. SEAP was also measurable in the sera from adult animals. This transgenic mouse will be a useful model for studying the signals that induce islet cell genesis in primary cultured tissues or intact animals.

ADA Funded Research

1607-P

Remission of Type 1 Diabetes by Adenoviral Vector-Mediated Betacellulin Gene Therapy

SEUNGHIN SHIN, JUN HEE-SOOK, YOON JI-WON. Calgary, AB, Canada; North Chicago, IL

Type 1 diabetes results from the loss of insulin-producing pancreatic [beta] cells. Human betacellulin (hBTC), a member of the epidermal growth factor family, has been shown to promote proliferation and/or differentiation of pancreatic islet cells. Treatment of diabetic mice with hBTC protein lowered blood glucose levels but did not normalize blood glucose, probably due to the short half-life of hBTC. This investigation was initiated to determine whether the continuous expression and secretion of hBTC can remit diabetes. We constructed a recombinant adenovirus (rAd- CMV-BTC) expressing hBTC under the cytomegalovirus promoter, and the [beta]-globin/IgG chimeric intron and albumin leader sequence was added upstream of the hBTC cDNA to enhance the secretion of hBT