Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver

The ability to identify, isolate, and transplant progenitor cells from solid tissues would greatly facilitate the treatment of diseases currently requiring whole organ transplantation. In this study, cell fractions enriched in candidate epithelial progenitor cells from the rat pancreas were isolated and transplanted into the liver of an inbred strain of Fischer rats. Using a dipeptidyl dipeptidase IV genetic marker system to follow the fate of transplanted cells in conjunction with albumin gene expression, we provide conclusive evidence that, after transplantation to the liver, epithelial progenitor cells from the pancreas differentiate into hepatocytes, express liver-specific proteins, and become fully integrated into the liver parenchymal structure. These studies demonstrate the presence of multipotent progenitor cells in the adult pancreas and establish a role for the liver microenvironment in the terminal differentiation of epithelial cells of foregut origin. They further suggest that such progenitor cells might be useful in studies of organ repopulation following acute or chronic liver injury.

Pax6 is essential for establishing ventral-dorsal cell boundaries in pituitary gland development

Pax6, a highly conserved member of the paired homeodomain transcription factor family that plays essential roles in ocular, neural, and pancreatic development and effects asymmetric transient dorsal expression during pituitary development, with its expression extinguished before the ventral → dorsal appearance of specific cell types. Analysis of pituitary development in the Small eye and Pax6 −/− mouse mutants reveals that the dorsoventral axis of the pituitary gland becomes ventralized, with dorsal extension of the transcriptional determinants of ventral cell types, particularly PFrk. This ventralization is followed by a marked decrease in terminally differentiated dorsal somatotrope and lactotrope cell types and a marked increase in the expression of markers of the ventral thyrotrope cells and SF-1-expressing cells of gonadotrope lineage. We suggest that the transient dorsal expression of Pax6 is essential for establishing a sharp boundary between dorsal and ventral cell types, based on the inhibition of Shh ventral signals.

Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be ≈30 μM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be ≈7 μM. Likewise, the NAD(P)H increase of the mitochondrial domain is ≈60 μM and is delayed with respect to the change in cytoplasmic NAD(P)H by ≈20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.

Purification of the β-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells

The β cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in β cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated β cells. The human cDNA coding for GSF/IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line αTC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed βTC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF/IPF-1, which may be present in limiting amounts in normal as opposed to transformed β cells.

Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes

One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

A mouse CD8 T cell-mediated acute autoimmune diabetes independent of the perforin and Fas cytotoxic pathways: Possible role of membrane TNF

Double transgenic mice [rat insulin promoter (RIP)-tumor necrosis factor (TNF) and RIP-CD80] whose pancreatic β cells release TNF and bear CD80 all develop an acute early (6 wk) and lethal diabetes mediated by CD8 T cells. The first ultrastructural changes observed in β cells, so far unreported, are focal lesions of endoplasmic reticulum swelling at the points of contact with islet-infiltrating lymphoblasts, followed by cytoplasmic, but not nuclear, apoptosis. Such double transgenic mice were made defective in either the perforin, Fas, or TNF pathways. Remarkably, diabetes was found to be totally independent of perforin and Fas. Mice lacking TNF receptor (TNFR) II had no or late diabetes, but only a minority had severe insulitis. Mice lacking the TNF-lymphotoxin (LTα) locus (whose sole source of TNF are the β cells) all had insulitis comparable to that of nondefective mice, but no diabetes or a retarded and milder form, with lesions suggesting different mechanisms of injury. Because both TNFR II and TNF-LTα mutations have complex effects on the immune system, these data do not formally incriminate membrane TNF as the major T cell mediator of this acute autoimmune diabetes; nevertheless, in the absence of involvement of the perforin or Fas cytotoxic pathways, membrane TNF appears to be the likeliest candidate.

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1.

Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371, 606-609]. In adults, IPF1 expression is restricted to the beta-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of beta-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet alpha-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta 2/NeuroD, which is expressed in both alpha- and beta-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-beta islet cells, which suggests an important role of IPF1 in beta-cell maturation.

Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA.

We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers.

Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode.

Amperometry at a carbon fiber microelectrode modified with a composite of ruthenium oxide and cyanoruthenate was used to monitor chemical secretions of single pancreatic beta cells from rats and humans. When the insulin secretagogues glucose, tolbutamide, and K+ were applied to the cell, a series of randomly occurring current spikes was observed. The current spikes were shown to be due to the detection of chemical substances secreted from the cell. Chromatography showed that the primary secreted substance detected by the electrode was insulin. The current spikes were strongly dependent on external Ca2+, had an average area that was independent of the stimulation method, and had an area distribution which corresponded to the distribution of vesicle sizes in beta cells. It was concluded that the spikes were due to the detection of concentration pulses of insulin secreted by exocytosis.

Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes.

The adenovirus (Ad) early region 3 (E3) genes code for at least four proteins that inhibit the host immune responses mediated by cytotoxic T lymphocytes and tumor necrosis factor alpha. To evaluate the potential use of these immunoregulatory viral functions in facilitating allogeneic cell transplantation, the Ad E3 genes were expressed in pancreatic beta cells in transgenic mice under control of the rat insulin II promoter. Transgenic H-2b/d (C57BL/6 x BALB/c) islets, expressing the Ad E3 genes, remained viable for at least 94 days after transplantation under the kidney capsule of BALB/c (H-2d) recipients. Nontransgenic H-2b/d control islets were rejected as anticipated between 14 and 28 days. Histological analysis of the transplanted transgenic islets revealed normal architecture. Immunohistochemical studies with antisera to islet hormones revealed the presence of both beta and non-beta islet cells, suggesting a propagation of the immunosuppressive effect of Ad proteins from beta cells to other islet cells. The use of viral genes, which have evolved to regulate virus-host interactions, to immunosupress the anti-genicity of donor transplant tissue suggests additional ways for prolonging allograft survival. In addition, these findings have implications for designing Ad vectors for gene therapy.

TERT promoter mutations in pancreatic endocrine tumours are rare and mainly found in tumours from patients with hereditary syndromes

One of the hallmarks of cancer is its unlimited replicative potential that needs a compensatory mechanism for the consequential telomere erosion. Telomerase promoter (TERTp) mutations were recently reported as a novel mechanism for telomerase re-activation/expression in order to maintain telomere length. Pancreatic endocrine tumors (PETs) were so far recognized to rely mainly on the alternative lengthening of telomeres (ALT) mechanism. It was our objective to study if TERTp mutations were present in pancreatic endocrine tumors (PET) and could represent an alternative mechanism to ALT. TERTp mutations were detected in 7% of the cases studied and were mainly associated to patients harbouring hereditary syndromes. In vitro, using PET-derived cell lines and by luciferase reporter assay, these mutations confer a 2 to 4-fold increase in telomerase transcription activity. These novel alterations are able to recruit ETS transcription factor members, in particular GABP-α and ETV1, to the newly generated binding sites. We report for the first time TERTp mutations in PETs and PET-derived cell lines. Additionally, our data indicate that these mutations serve as an alternative mechanism and in an exclusive manner to ALT, in particular in patients with hereditary syndromes.

Conditional inactivation of the Men1 gene leads to pancreatic and pituitary tumorigenesis but does not affect normal development of these tissues

Mutations of the MEN1 gene, encoding the tumor suppressor menin, predispose individuals to the cancer syndrome multiple endocrine neoplasia type 1, characterized by the development of tumors of the endocrine pancreas and anterior pituitary and parathyroid glands. We have targeted the murine Men1 gene by using Cre recombinase-loxP technology to develop both total and tissue-specific knockouts of the gene. Conditional homozygous inactivation of the Men1 gene in the pituitary gland and endocrine pancreas bypasses the embryonic lethality associated with a constitutional Men1(-/-) genotype and leads to beta-cell hyperplasia in less than 4 months and insulinomas and prolactinomas starting at 9 months. The pituitary gland and pancreas develop normally in the conditional absence of menin, but loss of this transcriptional cofactor is sufficient to cause beta-cell hyperplasia in some islets; however, such loss is not sufficient to initiate pituitary gland tumorigenesis, suggesting that additional genetic events are necessary for the latter.

PKC alpha is activated but not required during glucose-induced insulin secretion from rat pancreatic islets

The role of protein kinase C (PKC) in glucose-stimulated insulin secretion (GSIS) is controversial. Using recombinant adenoviruses for overexpression of PKCalpha and PKCdelta, in both wild-type (WT) and kinase-dead (KD) forms, we here demonstrate that activation of these two PKCs is neither necessary nor sufficient for GSIS from batch-incubated, rat pancreatic islets. In contrast, responses to the pharmacologic activator 12-O-tetradecanoylphorbol-13-acetate (TPA) were reciprocally modulated by overexpression of the PKCalphaWT or PKCalphaKD but not the corresponding PKCdelta adenoviruses. The kinetics of the secretory response to glucose (monitored by perifusion) were not altered in either cultured islets overexpressing PKCalphaKD or freshly isolated islets stimulated in the presence of the conventional PKC (cPKC) inhibitor Go6976. However, the latter did inhibit the secretory response to TPA. Using phosphorylation state-specific antisera for consensus PKC phosphorylation sites, we also showed that (compared with TPA) glucose causes only a modest and transient functional activation of PKC (maximal at 2-5 min). However, glucose did promote a prolonged (15 min) phosphorylation of PKC substrates in the presence of the phosphatase inhibitor okadaic acid. Overall, the results demonstrate that glucose does stimulate PKCalphain pancreatic islets but that this makes little overall contribution to GSIS.

Expression analysis of a human hepatic cell line in response to palmitate

Saturated fat plays a role in common debilitating diseases such as obesity, type 2 diabetes, and coronary heart disease. It is also clear that certain fatty acids act as regulators of metabolism via both direct and indirect signalling of target tissues. As the molecular mechanisms of saturated fatty acid signalling in the liver are poorly defined, hepatic gene expression analysis was undertaken in a human hepatocyte cell line after incubation with palmitate. Profiling of mRNA expression using cDNA microarray analysis revealed that 162 of approximately 18,000 genes tested were differentially expressed after incubation with palmitate for 48 h. Altered transcription profiles were observed in a wide variety of genes, including genes involved in lipid and cholesterol transport, cholesterol catabolism, cell growth and proliferation, cell signalling, P-oxidation, and oxidative stress response. While palinitate signalling has been examined in pancreatic beta-cells, this is the first report showing that palmitate regulates expression of numerous genes via direct molecular signalling mechanisms in liver cells. (C) 2005 Elsevier Inc. All rights reserved.