Pancreas Duodenum Homeobox-1 Research Articles

The Expression Pattern of PDX-1, SHH, Patched and Gli-1 Is Associated with Pathological and Clinical Features in Human Pancreatic Cancer

Background and Aims: Pancreatic cancer cells have been shown to possess stem-cell-like properties, especially by reactivating embryonic transcription factors involved in tissue differentiation. We therefore investigated if and to what extent developmental genes of the human pancreas are expressed in pancreatic ductal adenocarcinomas and precursor lesions, pancreatic intraepithelial neoplasia (PanIN), and if this correlates or predicts response to treatment and overall survival. Material and Methods: Invasive ductal adenocarcinomas of the pancreas [UICC pT3pN0 (n = 13) vs. pT3pN1 (n = 25)] and tumors after neoadjuvant chemotherapy [5-fluorouracil (FU)/folic-acid and gemcitabine; UICC ypNO (n = 7) vs. ypN1 (n = 6)] resected between 1997 and 2003 were characterized histochemically and immunohistochemically [pancreas duodenum homeobox 1 (PDX-1), Sonic hedgehog protein (SHH), Patched (Ptc) and Gli-1]. Gene distribution was compared with morphological patterns of the pancreatic carcinoma and PanIN as well as with peritumorous reactions of normal pancreas. Results: The overall expression of PDX-1, SHH, Ptc and Gli-1 was low, but showed a distinctive and topographic linkage inside pancreatic carcinomas as well as inside PanlNs. Additionally, a topographic and significant association of these markers with nodal status (PDX-1, Ptc, Gli-1), tumor size (PDX-1, Gli-1) and R status (PDX-1) was found. After stratification with the strongest outcome predictor, grading, survival analysis revealed that Ptc expression in grade 2 and PDX-1 expression in grade 3 carcinomas are independent survival factors. Conclusions: Markers of pancreas development are reexpressed in invasive ductal adenocarcinomas and their expression is essentially associated with general clinical and pathological features such as survival Or nodal Status.

Mesenchymal Stem Cells Adopt β-Cell Fate Upon Diabetic Pancreatic Microenvironment

: This study observed whether mesenchymal stem cells (MSCs) adopt beta-cell fate upon diabetic microenvironment. : We transplanted male porcine MSCs to diabetic female pigs by directly injecting into pancreas. Recipients' sera and pancreatic tissue were analyzed to assess the therapeutic effect. Islets were collected from the sections using laser-capture microdissection. The RNAs from these specimens were extracted and analyzed for insulin and pancreas duodenum homeobox 1 messenger RNA (mRNA) expression. SRY gene was detected from the specimens. : Compared with untreated diabetic controls, blood glucose level decreased greatly in recipients from 18 days (15.44 +/- 0.31 mmol/L vs 16.66 +/- 0.11 mmol/L) and insulin increased from 14 days (0.048 +/- 0.006 U/L vs 0.030 +/- 0.004 U/L). Hematoxylin and eosin-stained sections demonstrated increased islets in recipients and few lymphocytes present. The newly formed islets were smaller than normal islets (47.2 mum +/- 19.6 vs 119.6 +/- 27.7 mum). Reverse transcription-polymerase chain reaction showed that microdissected cells expressed insulin and pancreas duodenum homeobox 1 mRNA (79.3% +/- 16.2% of control, 65.2% +/- 14.8% of control, respectively). Immunoreactivity showed that the transplanted MSCs expressed insulin. SRY gene and insulin mRNA double-positive cells were found in microdissected cells by fluorescence in situ hybridization. This study shows that MSCs could adopt beta-cell fate in diabetic pancreatic microenvironment without obvious immune rejections. Stem cell transplantation in orthotope is a promising therapy for diabetes.

Differentiation of COPAS-sorted non-endocrine pancreatic cells into insulin-positive cells in the mouse

The regenerative process in the pancreas is of particular interest, since insulin-producing beta cells are lost in diabetes. Differentiation of new beta cells from pancreatic non-endocrine cells has been reported in vivo and in vitro, a finding that implies the existence of pancreatic stem/progenitor cells. However, while tissue-specific stem cells are well documented in skin, intestine and testis, pancreatic stem cells have been elusive. We hypothesised that pancreatic stem/progenitor cells within the non-endocrine fraction could be a source of new islets in vitro. To test if there were such cells within the pancreas, we generated pancreatic cell aggregates from tissue remaining after islet isolation from mouse insulin promoter 1-green fluorescent protein (MIP-GFP) mice. To eliminate any contamination of insulin-positive cells, we deleted all GFP-positive aggregates using COPAS Select and cultured with Matrigel. Immunohistochemistry, quantitative real-time PCR and single-cell nested RT-PCR were performed to confirm formation of insulin-producing cells. The GFP-negative cells were expanded as monolayers and then differentiated into three-dimensional cystic structures. After 1 week of culture, GFP-positive cells were found as clusters or single cells. By quantitative real-time PCR, no insulin mRNA was detected immediately after COPAS sorting, but after differentiation insulin mRNA of the whole preparation was 1.91 +/- 0.31% that of purified MIP-GFP beta cells. All GFP-positive cells expressed insulin 1; most expressed insulin 2, pancreas duodenum homeobox-1 and cytokeratin 19 by single cell nested RT-PCR. Our data support the concept that within the exocrine (acinar and ductal) pancreas of the adult mouse there are cells that can give rise to insulin-positive cells in vitro.

Adult mouse intrahepatic biliary epithelial cells induced in vitro to become insulin-producing cells

Transdifferentiation of cells from a patient's own liver into pancreatic beta-cells could be useful for beta-cell replacement. We hypothesized that intrahepatic biliary epithelial cells (IHBECs) could become a new source of insulin-producing cells. IHBECs isolated from adult mice were expanded using our novel culture method termed, collagen-embedded floating culture method (CEFCM). With CEFCM, IHBECs formed three-dimensional ductal cysts and rapidly expanded their number by about 15-fold within 2 weeks. Over 90% of cells were positive for cytokeratin 7 and 19. At day 14, IHBECs were transfected with adenoviral (Ad)- pancreas duodenum homeobox 1 (Pdx-1), NeuroD or Pdx-1/VP16. After 7 additional days in serum- and insulin-free differentiation medium (DM), cell phenotypes were determined by RT-PCR, immunostaining and ELISA for insulin. In DM control IHBECs started to express some endocrine progenitor genes (Neurog3, NeuroD, Nkx6.1, and Pdx-1) but lacked insulin gene (Ins) mRNA. Transduced expression of PDX-1, NEUROD or PDX-1/VP16 led to expression of not only INS but also GLUT2 and prohormone convertase 1 and 2. About 3% of 4000 cells counted in PDX-1/VP16 transduced cultures stained strongly for C-peptide suggesting that a subpopulation may have the capacity for differentiation. Transduced cells released insulin (Ad-PDX-1 0.08+/-0.05, Ad-NEUROD 0.33+/-0.09, Ad-PDX-1/VP16 0.37+/-0.14 ng/1x10(5) cells after 48 h in culture). IHBECs can be markedly expanded, and then with molecular manipulation a subpopulation of these cells can differentiate towards a beta-cell phenotype. This approach may lead to a new source of beta-cells that can be used for transplantation in diabetes.

The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells

Islet-like cells derived from embryonic stem (ES) cells may be a promising therapeutic option for future diabetes treatment. Here, we demonstrated a five-stage protocol with adding exendin-4 instead of nicotinamide finally could generate islet-like cells from human embryonic stem (ES) cells. Immunofluorescence analysis revealed a high percentage of c-peptide positive cells in the derivation. However, in addition to insulin/c-peptide, most cells also coexpressed PDX-1 (pancreas duodenum homeobox-1), glucagon, somatostatin or pancreatic polypeptide. Insulin and other pancreatic beta-cell-specific genes were all present in the differentiated cells. Insulin secretion could be detected and increased significantly by adding KCL in high glucose concentration in vitro. Furthermore, subcutaneous transplantation of scaffolds seeded with the islet-like cells or cell transplantation under kidney capsules for further differentiation in vivo could improve 6 h fasted blood glucose levels and diabetic phenotypes in streptozotocin-induced diabetic SCID mice. More interestingly, blood vessels of host origin, characterized by mouse CD31 immunostaining, invaded the cell–scaffold complexes. This work reveals a five-stage protocol with adding exendin-4 may be an effective protocol on the differentiation of human ES cells into islet-like cells, and suggests scaffolds can serve as vehicles for islet-like cell transplantation.

Three-dimensional Cultures of Rat Pancreatic RIN-5F Cells in Porous PLGA-collagen Hybrid Scaffolds

Three-dimensional cultures of pancreatic islet cells in porous scaffolds or hydrogels have been constructed as a biohybrid artificial pancreas. A thin mesh of a PLGA-collagen hybrid was used to culture rat RIN-5F cells. The hybrid mesh was coated with laminin, fibronectin, vitronectin, type IV collagen, and poly(L-lysine) were evaluated and mesh without coating was used as a control. Cell adhered and proliferated on all of the coated and uncoated meshes. The cells formed spheroids in the uncoated, poly(L-lysine)-, fibronectin-, vitronectin-, and type IV collagen-coated hybrid meshes, while forming a layered structure in the laminin-coated hybrid mesh. Cell adhesion on the coated PLGA-collagen hybrid meshes was higher than that for the uncoated hybrid mesh. The laminin-coated hybrid mesh showed the greatest level of adhesion. The insulin secretion capacity of RIN-5F cells was at the same level for all coated and uncoated PLGA-collagen hybrid meshes and higher than that of cells cultured on cell culture plates. The 3D cultured PLGA-collagen hybrid meshes promoted insulin production capacity. Gene expression analysis showed that genes encoding insulin I, insulin II, and the pancreatic transcription factor PDX-1 (pancreas/duodenum homeobox 1) was expressed. These results indicate that the PLGA-collagen hybrid meshes support adhesion, proliferation, and differentiation of RIN-5F cells that allows culturing pancreatic islet cells on 3D constructs.

Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells

Pancreatic islet transplantation has demonstrated that long-term insulin independence may be achieved in patients suffering from diabetes mellitus type 1. However, limited availability of islet tissue means that new sources of insulin-producing cells that are responsive to glucose are required. Here, we show that human amnion epithelial cells (HAEC) can be induced to differentiate into functional insulin-producing cells in vitro. After induction of differentiation, HAEC expressed multiple pancreatic β-cell genes, including insulin, pancreas duodenum homeobox-1, paired box gene 6, NK2 transcription factor-related locus 2, Islet 1, glucokinase, and glucose transporter-2, and released C-peptide in a glucose-regulated manner in response to other extracellular stimulations. The transplantation of induced HAEC into streptozotocin-induced diabetic C57 mice reversed hyperglycemia, restored body weight, and maintained euglycemia for 30 d. These findings indicated that HAEC may be a new source for cell replacement therapy in type 1 diabetes.

Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells

Pancreatic islet transplantation has demonstrated that long-term insulin independence may be achieved in patients suffering from diabetes mellitus type 1. However, limited availability of islet tissue means that new sources of insulin-producing cells that are responsive to glucose are required. Here, we show that human amnion epithelial cells (HAEC) can be induced to differentiate into functional insulin-producing cells in vitro. After induction of differentiation, HAEC expressed multiple pancreatic beta-cell genes, including insulin, pancreas duodenum homeobox-1, paired box gene 6, NK2 transcription factor-related locus 2, Islet 1, glucokinase, and glucose transporter-2, and released C-peptide in a glucose-regulated manner in response to other extracellular stimulations. The transplantation of induced HAEC into streptozotocin-induced diabetic C57 mice reversed hyperglycemia, restored body weight, and maintained euglycemia for 30 d. These findings indicated that HAEC may be a new source for cell replacement therapy in type 1 diabetes.

Taurine supplementation modulates glucose homeostasis and islet function

Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca 2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5·6 and 11·1-mmol/L concentrations; (v) slowed cytosolic Ca 2+ concentration ([Ca 2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity.

Mutated ATP synthase induces oxidative stress and impaired insulin secretion in β‐cells of female BHE/cdb rats

Adenosine triphosphate (ATP) is a critical determinant of beta-cell insulin secretion in response to glucose. BHE/cdb rats have a mutation in ATP synthase that limits ATP production, yet develop mild diabetes only with ageing. We investigated the cellular basis for reduced insulin secretion and compensatory mechanisms that mitigate the effects of the ATP synthase mutation. In vitro beta-cell function in isolated islets and expression of key regulatory genes was compared with in vivo oral glucose tolerance and insulin sensitivity in BHE/cdb and control rats. BHE/cdb rat islets had reduced responsiveness to glucose stimulation and ATP content was 35% lower than in control islets. Oral glucose tolerance was impaired at both 21 and 43 weeks of age because of a reduction in glucose-stimulated insulin secretion (GSIS). An increase in inducible nitric oxide synthase (INOS, 3-fold) and manganese superoxide dismutase (MnSOD, 1.6-fold), detection of nitrotyrosine, beta-cell apoptosis, and nucleocytoplasmic translocation of pancreas duodenum homeobox-1 (PDX-1) in beta-cells indicated increased oxygen radical formation. However, BHE/cdb rats partially compensated for low glucose responsiveness by increasing the number of small islets and beta-cell hypertrophy. There was also an increase in the proportion of mature insulin relative to proinsulin (PI) detected within beta-cell granules. Increased activation of AMP-dependent kinase (AMPK)-regulated pathways was consistent with increased oxidative stress and with induction of apoptosis and reduction of preproinsulin gene transcription. The findings are consistent with impaired but partially compensated mechanisms of insulin secretion early in life, but progressive non-compensated impairments due to oxidative stress occurs by age 43 weeks.

Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic β-cells

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic β-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic β-cells, two Nnat isotypes (α and β) were expressed using adenoviruses in murine MIN6N8 pancreatic β-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatα and Nnatβ were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatβ to Nnatα ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatβ was inclined to form aggresome-like structures, and we found that Nnatβ aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatβ sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatβ increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatβ under high glucose conditions decreased the expression of genes important for β-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal β-cell function, as well as in the pathogenesis of type 2 diabetes.

Exendin-4 and exercise promotes β-cell function and mass through IRS2 induction in islets of diabetic rats

Not only exendin-4 but also exercise has been reported to improve glucose homeostasis by enhancing insulinotropic action, but the nature of its molecular mechanism has not been clarified. We investigated a mechanism to promote insulinotropic action by means of exendin-4 and exercise training in 90% pancreatectomized (Px) rats fed 40% energy fat diets. Px diabetic rats were divided into 4 groups: 1) exendin-4, 2) exendin-4 plus exercise, 3) saline (control), and 4) exercise. During the 8-week experimental period, rats in the exendin-4 groups were subcutaneously administered with 150 pmol/kg exendin-4 twice a day, while those in the exercise groups ran on an uphill treadmill with a 15 degree incline at 20 m/min for 30 min 5 days a week. First phase insulin secretion was elevated by both the administration of exendin-4 and exercise training during hyperglycemic clamp. However, second phase insulin secretion did not differ among the groups. Individual treatment of exendin-4 and exercise expanded β-cell mass by increasing its proliferation and reducing its apoptosis, but the administration of exendin-4 plus exercise training did not produce any additional, positive effects. Both exendin-4 and exercise enhanced insulin receptor substrate (IRS)-2 expression through the activation of cAMP responding element binding protein in the islets, which potentiated their insulin/insulin like growth factor-1 signaling. The potentiation of the signaling increased the expression of pancreas duodenum homeobox-1, involved in β-cell proliferation. In conclusion, exendin-4 and exercise equivalently improved glucose homeostasis due to the induction of IRS-2 in the islets of diabetic rats through a cAMP dependent common pathway.

Extracts of Rehmanniae radix, Ginseng radix and Scutellariae radix improve glucose-stimulated insulin secretion and β-cell proliferation through IRS2 induction

Recent studies have revealed that beta-cell dysfunction is an important factor in developing type 2 diabetes. beta-cell dysfunction is related to impairment of the insulin/IGF-1 signaling cascade through insulin receptor substrate-2 (IRS2). The induction of IRS2 in beta-cells plays an important role in potentiating beta-cell function and mass. In this study, we investigated whether herbs used for treating diabetes in Chinese medicine-Galla rhois, Rehmanniae radix, Machilus bark, Ginseng radix, Polygonatum radix, and Scutellariae radix-improved IRS2 induction in rat islets, glucose-stimulated insulin secretion and beta-cell survival. R. radix, Ginseng radix and S. radix significantly enhanced glucose-stimulated insulin secretion compared to the control, i.e., by 49, 67 and 58%, respectively. These herbs induced the expression of IRS2, pancreas duodenum homeobox-1 (PDX-1), and glucokinase. The increased level of glucokinase could explain the enhancement of glucose-stimulated insulin secretion with these extracts. Increased PDX-1 expression was associated with beta-cell proliferation, which was consistent with the cell viability assay. In conclusion, R. radix, Ginseng radix and S. radix had an insulinotropic action similar to that of exendin-4.

Beneficial effects of L‐arginine–nitric oxide‐producing pathway in rats treated with alloxan

In an attempt to elucidate molecular mechanisms and factors involved in beta cell regeneration, we evaluated a possible role of the L-arginine-nitric oxide (NO)-producing pathway in alloxan-induced diabetes mellitus. Diabetes was induced in male Mill Hill rats with a single alloxan dose (120 mg kg(-1)). Both non-diabetic and diabetic groups were additionally separated into three subgroups: (i) receiving L-arginine . HCl (2.25%), (ii) receiving L-NAME . HCl (0.01%) for 12 days as drinking liquids, and (iii) control. Treatment of diabetic animals started after diabetes induction (glucose level > or = 12 mmol l(-1)). We found that disturbed glucose homeostasis, i.e. blood insulin and glucose levels in diabetic rats was restored after L-arginine treatment. Immunohistochemical findings revealed that L-arginine had a favourable effect on beta cell neogenesis, i.e. it increased the area of insulin-immunopositive cells. Moreover, confocal microscopy showed colocalization of insulin and pancreas duodenum homeobox-1 (PDX-1) in both endocrine and exocrine pancreas. This increase in insulin-expressing cells was accompanied by increased cell proliferation (observed by proliferating cell nuclear antigen-PCNA immunopositivity) which occurred in a regulated manner since it was associated with increased apoptosis (detected by the TUNEL method). Furthermore, L-arginine enhanced both nuclear factor-kB (NF-kB) and neuronal nitric oxide synthase (nNOS) immunopositivities. The effect of L-arginine on antioxidative defence was observed especially in restoring to control level the diabetes-induced increase in glutathione peroxidase activity. In contrast to L-arginine, diabetic pancreas was not affected by L-NAME supplementation. In conclusion, the results suggest beneficial L-arginine effects on alloxan-induced diabetes resulting from the stimulation of beta cell neogenesis, including complex mechanisms of transcriptional and redox regulation.

Sterol regulatory element–binding protein‐1c and pancreatic β‐cell dysfunction

It has long been known that excess intracellular fatty acids cause impaired insulin secretion, referred to as beta-cell lipotoxicity. Sterol regulatory element-binding protein (SREBP)-1c is a transcription factor that controls hepatic fatty acid synthesis. Activation of SREBP-1c by overnutrition also inhibits insulin receptor substrate-2 (IRS-2) and induces insulin resistance in the liver. As SREBP-1c is also expressed in beta cells, we hypothesized that activation of SREBP-1c could be a part of the mechanism by which saturated fatty acids induce beta-cell lipotoxicity. We found that nuclear SREBP-1c has a negative impact on both glucose- and potassium-stimulated insulin secretion as determined in islets from beta-cell-specific SREBP-1c transgenic mice as well as SREBP-1c knockout mice. This effect of SREBP-1c involves multiple functional pathways required for insulin secretion from beta cells: (i) decreased ATP caused by energy consumption through lipogenesis and uncoupling protein-2 (UCP-2) activation; (ii) repressed IRS-2 and pancreas duodenum homeobox 1 (PDX1) expression, leading to impaired beta-cell mass; and (iii) impaired post-ATP membrane voltage-dependent steps of the insulin secretion pathway caused by upregulated granuphilin and other ion channel proteins. Saturated fatty acids, such as palmitic acid (PA), impair insulin secretion through SREBP-1c activation, whereas polyunsaturated fatty acids including eicosapentaenoic acid (EPA) restore PA-suppressed insulin secretion through suppression of SREBP-1c. These data implicate a therapeutic potential of EPA against insulin secretion defects caused by lipotoxicity.

Changes in Components, Glycyrrhizin and Glycyrrhetinic Acid, in RawGlycyrrhiza uralensisFisch, Modify Insulin Sensitizing and Insulinotropic Actions

We hypothesized that roasted Glycyrrhizae Radix (Glycyrrhizin Radix Praeparata, GRP) might modify anti-diabetic action due to compositional changes. Then we examined the anti-diabetic effect and mechanism of raw Glycyrrhizae Radix (GR) and GRP extracts and their major respective components, glycyrrhizin and glycyrrhetinic acid. In partial pancreatectomized (Px) diabetic mice, both GR and GRP improved glucose tolerance, but only GRP enhanced glucose-stimulated insulin secretion as much as exendin-4. Both GR and GRP extracts enhanced insulin-stimulated glucose uptake through peroxisome proliferation-activated receptor (PPAR)-gamma activation in 3T3-L1 adipocytes. Consistently with the results of the mice study, only GRP and glycyrrhetinic acid enhanced glucose-stimulated insulin secretion in isolated islets. In addition, they induced mRNA levels of insulin receptor substrate-2, pancreas duodenum homeobox-1, and glucokinase in the islets, which contributed to improving beta-cell viability. In conclusion, GRP extract containing glycyrrhetinic acid improved glucose tolerance better than GR extract by enhancing insulinotropic action. Thus, GRP had better anti-diabetic action than GR.

Acinar Plasticity

The plasticity of pancreatic tissue is demonstrated in many pancreatic diseases. It has previously been shown that pancreatic islet-to-duct transformation and acinoductal metaplasia have been associated with both pancreatic regeneration and adenocarcinoma in various in vivo and in vitro settings. Understanding this inherent morphogenetic plasticity of the adult pancreas could lead to new therapeutic approaches to pancreatic disease. Cadaveric human pancreases (n = 7) were digested, and purified acinar tissue, which was approximately 85% immunoreactive for amylase and approximately 15% immunoreactive for CK-19, was embedded in a type 1 collagen matrix and cultured in a differentiation medium (DM) consisting of Dulbecco modified Eagle/F12 medium supplemented with cholera toxin (100 ng/mL), epidermal growth factor (10 ng/mL), and insulin (24 mU/mL) for 8 days. After this initial period, the resulting tissues were cultured in DM without cholera toxin, supplemented with gastrin (50 nmol/L) and hepatocyte growth factor (HGF; 10 ng/mL), with islet neogenesis-associated protein (INGAP; 167 nmol/L) or with gastrin + HGF + INGAP for 6 days. Tissue samples were then analyzed for amylase, cytokeratin 19, pancreas duodenum homeobox 1, and endocrine hormone immunoreactivity as well as dithizione positivity. After 8 days of culture, approximately 90% of acini transformed into ductlike structures. This acinoductal transformation was characterized by a complete absence of amylase staining, with virtually all cells CK-19 immunoreactive. Addition of INGAP led to an approximately 18-fold increase in pancreas duodenum homeobox 1 immunoreactivity, although without an observed increase in insulin production as measured by dithizone positivity. However, when acinar-derived ductlike structures were cultured with gastrin + HGF + INGAP, the total incidence of dithizone-positive structures increased approximately 6-fold (10.9 +/- 2.9% vs 1.7 +/- 0.4%, P = 0.037). Treatment with gastrin + HGF alone led to no significant change in any of the measured parameters. We have developed a novel in vitro model of adult human acinoductal metaplasia that will aid not only in developing new methods of expanding beta-cell mass but also provide insights into pancreatic carcinogenesis.

Regulation of Pancreas Duodenum Homeobox-1 Expression by Early Growth Response-1

The homeodomain transcription factor pancreas duodenum homeobox-1 (PDX-1) is a key regulator of pancreatic beta-cell development, function, and survival. Deficits in PDX-1 expression result in insulin deficiency and hyperglycemia. We previously found that the glucose-responsive transcription factor early growth response-1 (Egr-1) activates the insulin promoter in part by increasing expression levels of PDX-1. We now report that Egr-1 binds and activates multiple regulatory sites within the pdx-1 promoter. We identified consensus Egr-1 recognition sequences within proximal and distal regions of the mouse pdx-1 promoter and demonstrated specific binding of Egr-1 by chromatin immunoprecipitation and electrophoretic mobility shift assays. Overexpression of Egr-1 increased transcriptional activation of the -4500 proximal pdx-1 promoter and of the highly conserved regulatory Areas I, II, and III. Mutagenesis of a specific Egr-1 binding site within Area III substantially decreased Egr-1-mediated activation. Egr-1 increased the transcriptional activation of Areas I and II, despite the absence of Egr-1 recognition sequences within this promoter segment, suggesting that Egr-1 also can regulate the pdx-1 promoter indirectly. Egr-1 increased, and a dominant-negative Egr-1 mutant repressed, the transcriptional activation of distal pdx-1 promoter sequences. Mutagenesis of a specific Egr-1 binding site within regulatory Area IV reduced basal and Egr-1-mediated transcriptional activation. Our data indicate that Egr-1 regulates expression of PDX-1 in pancreatic beta-cells by both direct and indirect activation of the pdx-1 promoter. We propose that Egr-1 expression levels may act as a sensor in pancreatic beta-cells to translate extracellular signals into changes in PDX-1 expression levels and pancreatic beta-cell function.

Negative Regulation of c-Myc Transcription by Pancreas Duodenum Homeobox-1

The pancreatic and duodenal homeobox factor-1 (Pdx1) is essential for pancreatic development and insulin gene transcription, whereas c-Myc has a deleterious effect on islet function. However, the relationship between c-Myc and Pdx1 is poorly concerned. Here we demonstrated that Pdx1 could suppress c-Myc promoter activity, which relied on T cell factor (Tcf) binding elements harbored in c-Myc promoter. Furthermore, the transcription activity of beta-catenin/Tcf was markedly decreased on Pdx1 expression, but cotransfection of Pdx1 short hairpin RNA abrogated this effect. Pdx1 expression did not induce beta-catenin degradation nor did it alter their subcellular distribution. The mutation analysis showed that the amino acids (1-209) of Pdx1 harboring an inhibitory domain, which might lead to the reduction of beta-catenin/Tcf/p300 complex levels and attenuate their binding activity with c-Myc promoter sequences. Moreover, adenovirus-mediated Pdx1 interference caused cell proliferation and cytokine-induced apoptosis via the dysregulation of c-Myc transcription. These results indicated that the Pdx1 functioned as a key regulator for maintenance of beta-cell function, at least in part, through controlling c-Myc expression and the loss of its regulatory function may be an alternative mechanism for beta-cell neogenesis and apoptosis found in diabetes.

Impaired insulin secretion in vivo but enhanced insulin secretion from isolated islets in pancreatic beta cell-specific vascular endothelial growth factor-A knock-out mice

Endothelial cells are considered to be essential for normal pancreatic beta cell function. However, there have been no reports showing their importance for beta cell function. Using mice with disrupted vascular endothelial growth factor-A gene specifically in beta cells, we investigated the relation between islet vascular structure and beta cell function. Mice with disrupted vascular endothelial growth factor-A gene specifically in beta cells had reduced islet vascular density with impaired formation of endothelial fenestration. While their fasting glucose and body weight were comparable with control mice, their glucose- and tolbutamide-induced rapid insulin release were impaired, thus resulting in glucose intolerance. On the other hand, glucose and KCl enhanced the levels of insulin secreted from islets isolated from these mice. In addition, the production of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in the islets was increased. Insulin content and expression of insulin I and pancreas duodenum homeobox 1 mRNA in the islets were also increased. Our results indicate that an abnormal quality and quantity of blood vessels due to reduced expression of vascular endothelial growth factor-A in beta cells could be a cause of impaired insulin secretion without impairment of beta cell function.