Kevin Docherty

Pancreatic duodenal homeobox–1, PDX-1, a major regulator of beta cell identity and function

Abstract. Pancreatic duodenal homeobox –1 is a transcription factor that is expressed in beta and δ cells of the islets of Langerhans and in dispersed endocrine cells of the duodenum. It is involved in regulating the expression of a number of key beta-cell genes as well as somatostatin. It also plays a pivotal part in the development of the pancreas and islet cell ontogeny. Thus homozygous disruption of the gene in mice and humans results in pancreatic agenesis. Heterozygous mutations in the gene result in impaired glucose tolerance and symptoms of diabetes as seen in MODY4 and late-onset Type II (non-insulin-dependent) diabetes mellitus. In adults pancreatic duodenal homeobox-1 expression is increased in duct cells of the pancreas that have been induced to proliferate and differentiate to form new islets. Defects in pancreatic duodenal homeobox-1 could therefore contribute to Type II diabetes by affecting compensatory mechanisms that increase the rate of beta-cell neogenesis to meet the increased insulin secretory demand. It could also be a pharmacological target for beta-cell defects in Type II diabetes, while its role as a regulator of islet stem cell activity is being exploited to produce a replenishable source of islet tissue for transplantation in Type I (insulin-dependent) diabetes mellitus. [Diabetologia (2001) 44: 1203–1214]

The Insulin Gene Promoter: A Simplified Nomenclature

The tools of molecular biology have rapidly expanded our knowledge of how β-cells regulate insulin gene expression. As this work has progressed in parallel in different laboratories, alternate nomenclature systems have been developed to describe the functionally important elements of the insulin gene. This jumble of names is confusing to those outside the field and intimidating to neophytes. Therefore, we have agreed to a simple, uniform set of names for the major insulin gene promoter elements.

Pancreatic transcription factors and their role in the birth, life and survival of the pancreatic β cell

In recent years major progress has been made in understanding the role of transcription factors in the development of the endocrine pancreas in the mouse. Here we describe how a number of these transcription factors play a role in maintaining the differentiated phenotype of the beta cell, and in the mechanisms that allow the beta cell to adapt to changing metabolic demands that occur throughout life. Amongst these factors, Pdx1 plays a critical role in defining the region of the primitive gut that will form the pancreas, Ngn3 expression drives cells towards an endocrine lineage, and a number of additional proteins including Pdx1, in a second wave of expression, Pax4, NeuroD1/beta2, and MafA act as beta cell differentiation factors. In the mature beta cell Pdx1, MafA, beta2, and Nkx2.2 play important roles in regulating expression of insulin and to some extent other genes responsible for maintaining beta cell function. We emphasise here that data from gene expression studies in rodents seldom map on to the known structure of the corresponding human promoters. In the adult the beta cell is particularly susceptible to autoimmune-mediated attack and to the toxic metabolic milieu associated with over-eating, and utilises a number of these transcription factors in its defence. Pdx1 has anti-apoptotic and proliferative activities that help facilitate the maintenance of beta cell mass, while Ngn3 may be involved in the recruitment of progenitor cells, and Pax4 (and possibly HNF1alpha and Hnf4alpha) in the proliferation of beta cells in the adult pancreas. Other transcription factors with a more widespread pattern of expression that play a role in beta cell survival or proliferation include Foxo1, CREB family members, NFAT, FoxM1, Snail and Asc-2.

Relative contribution of PDX-1, MafA and E47/β2 to the regulation of the human insulin promoter

The insulin promoter binds a number of tissue-specific and ubiquitous transcription factors. Of these, the homoeodomain protein PDX-1 (pancreatic duodenal homeobox factor-1), the basic leucine zipper protein MafA and the basic helix-loop-helix heterodimer E47/BETA2 (beta-cell E box transactivator 2; referred to here as beta2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta2 to activate the rat insulin 1 promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/beta2 in regulating the human insulin promoter, and whether these factors could interact synergistically in the context of the human promoter. Mutagenesis of the PDX-1, MafA and E47/beta2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 beta-cells. In the islet glucagonoma cell line alphaTC1.6, overexpression of PDX-1 and MafA separately increased promoter activity approx. 2.5-3-fold, and in combination approx. 6-fold, indicating that their overall effect was additive. Overexpression of E47 and beta2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and beta2 each increased activity by less than 2-fold. There was no indication of any synergistic effects on the human insulin promoter. On the other hand, the rat insulin 1 promoter and a mutated version of the human insulin promoter, in which the relevant regulatory elements were separated by the same distances as in the rat insulin 1 promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin 1 gene in alphaTC1.6 cells, whereas MafA activated the insulin 2 gene. In combination, PDX-1 and MafA activated both insulin genes. Chromatin immunoprecipitation assays confirmed that PDX-1 increased the association of acetylated histones H3 and H4 with the insulin 1 gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.

PROCESSING OF PRO-ISLET AMYLOID POLYPEPTIDE (PROIAPP) BY THE PROHORMONE CONVERTASE PC2

Islet amyloid polypeptide (IAPP), ‘amylin’, is the component peptide of islet amyloid formed in Type 2 diabetes. IAPP is expressed in islet β‐cells and is derived from a larger precursor, proIAPP, by proteolysis. An in vitro translation/translocation system was used to separately examine processing of human proIAPP by the β‐cell endopeptidases PC2, PC3 or furin. ProIAPP was converted to mature IAPP by PC2 but there was little conversion by furin or PC3. These data are consistent with processing of proIAPP in β‐cell secretory granules. Abnormal cellular proteolysis associated with type 2 diabetes could contribute to IAPP amyloidosis.

Biphasic Induction of Pdx1 in Mouse and Human Embryonic Stem Cells Can Mimic Development of Pancreatic β‐Cells

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic β‐cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell. By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage‐dependent requirement for this transcription factors activity. Mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas‐like cell phenotype, in which 30% of the cells were β‐cell‐like. Cell markers consistent with the different β‐cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development. Furthermore, in mouse ES‐derived cultures the differentiated β‐like cells secreted C‐peptide (insulin) in response to KCl and 3‐isobutyl‐1‐methylxanthine, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells. Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non‐β‐cells in the developing endocrine pancreas. Collectively, we show that this method of in vitro differentiation provides a template for inducing and studying ES cell differentiation into insulin‐secreting cells. STEM CELLS 2009;27:341–351

γ-Aminobutyric Acid Up-Regulates the Expression of a Novel Secretogranin-II Messenger Ribonucleic Acid in the Goldfish Pituitary1

An RNA-arbitrarily primed PCR differential display strategy was used to identify candidate genes in the pituitary that are up-regulated by endogenously activated g-aminobutyric acid (GABA) systems that may also be involved in the control of reproduction. Goldfish were injected with the GABA metabolism inhibitor g-vinyl-GABA (GVG), known for its high efficiency to specifically increase endogenous brain and pituitary GABA levels in this species, resulting in higher levels of circulating gonadotropin-II (GTH-II). Several transcripts related to hormone secretion, signal transduction pathways, and messenger RNA (mRNA) editing were shown to be up-regulated after GVG injection. Among these transcripts we characterized an mRNA coding for the secretory vesicle protein secretogranin-II (SgII), a member of the chromogranin family, which is the precursor of a novel 34 amino acid neuropeptide, goldfish secretoneurin (SN). A semiquantitative PCR developed to measure pituitary SgII mRNA levels showed a 5-fold increase in GVG treated fish vs. control fish. Moreover, GVG treatment specifically increased SgII mRNA levels in gonadotrophs, concomitant with a decrease in GTH-II cell content. In addition, ip injection of synthetic goldfish SN increased GTH-II release in goldfish pretreated with the dopamine antagonist domperidone. Activation of GABAergic neurons has two effects, enhancing in vivo GTH-II release and up-regulating SgII mRNA specifically in goldfish gonadotrophs. Together with our SN bioactivity data, this suggests the existence in the pituitary of an autocrine or paracrine mechanism linked to the regulated secretory pathway in the gonadotrophs. (Endocrinology 139: 4870–4880, 1998) T AMINO ACID g-aminobutyric acid (GABA) is one of the most abundant neurotransmitters in the vertebrate central nervous system and is considered to be a classical inhibitory neurotransmitter inducing postsynaptic membrane hyperpolarizations. However, in addition to the predominant inhibitory actions reported for GABA, there is increasing evidence in both vertebrates (1) and invertebrates (2) that GABA also has important depolarizing and stimulatory actions. In the rat hypothalamus, for example, GABA can be found in approximately 50% of presynaptic boutons (3) and regulates most aspects of hypothalamic function. In particular, GABA via the GABAA receptor is excitatory in neonatal hypothalamic neurons, whereas in adults the opposite has been shown (4). Recently, dual hyperpolarizing and depolarizing actions of GABA have been shown in the adult rat suprachiasmatic nucleus of the hypothalamus (5). Within this nucleus, GABA is inhibitory at night but has important excitatory actions in the daytime, which may be part of the molecular mechanism of the circadian clock, coordinating diurnal changes in behavior and physiology. In adult vertebrates, GABAergic control of neuroendocrine function is also believed by many to be mainly inhibitory. However, a significant stimulatory role for GABA in the control of hypothalamic GnRH (GnRH) (6) and pituitary gonadotropic hormone (7, 8) release is now apparent. Our work using the adult goldfish model (reviewed in Ref. 9) has demonstrated that GABA has a clear and potent stimulatory effect on pituitary gonadotropin-II (GTH-II; the fish homolog of LH), which in turn stimulates reproductive function (i.e. gonadal sex steroid production, ovulation, or sperm release). The goldfish, as in other bony fish, lack a hypothalamopituitary portal system and the proximal pars distalis, that part of the anterior pituitary containing the gonadotroph and somatotroph cells, is directly innervated by a multitude of neurons synthesizing neuropeptides and classical neurotransmitters (10), including GABA (11). Moreover, because nerve terminals reside within the anterior pituitary complex, this is a unique system in which to study neurotransmitterendocrine cell interactions (10). Significantly, the magnitude of the GTH-II secretory response is often greater to GABA than to any other peptide or neurotransmitter acting on the system, indicating that GABA is a pivotal neurotransmitter for central reproductive control. In vivo, pharmacological evidence indicates that GABA action to enhance GTH-II release is mediated by dual stimulatory effects on GABAA and GABAB receptors (8), which may explain why GABA has a dominant stimulatory effect in the goldfish model. GABA acts to stimulate GTH-II release Received April 27, 1998. Address all correspondence and requests for reprints to: Vance L. Trudeau, Department of Biology, University of Ottawa. P.O. Box 450, Station A. Ottawa, Ontario K1N 6N5, Canada. E-mail: vtrudeau@ science.uottawa.ca. * The work was supported by the Welcome Trust (UK), BalaguerGonell Foundation (Spain), and Natural Sciences and Engineering Research Council (Canada). 0013-7227/98/

Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells

03.00/0 Vol. 139, No. 12 Endocrinology Printed in U.S.A. Copyright

Suppression of Epithelial-to-Mesenchymal Transitioning Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue Toward Functional Insulin-Producing β-Like Cells

Aims/hypothesisHuman embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge.MethodsWe used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors. This approach avoids the use of feeders, stroma cells or serum, all of which can interfere with experimental outcomes and could preclude future clinical applications.ResultsThis study reveals, for the first time, that activin/TGF-β signalling blocks pancreatic specification induced by retinoic acid while promoting hepatic specification in combination with bone morphogenetic protein and fibroblast growth factor. Using this knowledge, we developed culture systems to differentiate human pluripotent stem cells into near homogenous population of pancreatic and hepatic progenitors displaying functional characteristics specific to their natural counterparts. Finally, functional experiments showed that activin/TGF-β signalling achieves this essential function by controlling the levels of transcription factors necessary for liver and pancreatic development, such as HEX and HLXB9.Conclusion/interpretationOur methods of differentiation provide an advantageous system to model early human endoderm development in vitro, and also represent an important step towards the generation of pancreatic and hepatic cells for clinical applications.

Impaired expression of transcription factor IUF1 in a pancreatic β-cell line derived from a patient with persistent hyperinsulinaemic hypoglycaemia of infancy (nesidioblastosis)

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.