[Inherited alopecia X in Pomeranians]

Four male Pomeranians that showed alopecia with an age of onset between five months and eight years were investigated.The aim of the investigation was to clarify whether the affected dogs had alopecia X and whether their symptoms might be due to a hereditary defect.The four affected dogs showed hairless patches at the root of the tail, at the back, at the limbs from the thigh to the tarsus and at the abdomen. Within the hairless patches some islets with sparse hair were present. In hairless patches the skin was dark pigmented. Besides the alopecia and hyperpigmentation no other symptoms were found according to anamnestic and clinical examination. History, clinical examinations, laboratory diagnostics, and histopathology of skin biopsies allowed the diagnosis of alopecia X in three affected male dogs.The last one of the affected dogs additionally had slightly reduced thyroid hormone levels. Based on identical symptoms and the close relatedness of all four animals, it was assumed that the fourth affected dog also had alopecia X.The available data possibly indicate a monogenic autosomal dominant inheritance, however a recessive inheritance can not be excluded at this time.

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Tmem27: a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation

The signals and molecular mechanisms that regulate the replication of terminally differentiated beta cells are unknown. Here, we report the identification and characterization of transmembrane protein 27 (Tmem27, collectrin) in pancreatic beta cells. Expression of Tmem27 is reduced in Tcf1(-/-) mice and is increased in islets of mouse models with hypertrophy of the endocrine pancreas. Tmem27 forms dimers and its extracellular domain is glycosylated, cleaved and shed from the plasma membrane of beta cells. This cleavage process is beta cell specific and does not occur in other cell types. Overexpression of full-length Tmem27, but not the truncated or soluble protein, leads to increased thymidine incorporation, whereas silencing of Tmem27 using RNAi results in a reduction of cell replication. Furthermore, transgenic mice with increased expression of Tmem27 in pancreatic beta cells exhibit increased beta cell mass. Our results identify a pancreatic beta cell transmembrane protein that regulates cell growth of pancreatic islets.

Reduced beta-cell mass and altered glucose sensing impair insulin-secretory function in betaIRKO mice

Pancreatic beta-cell-restricted knockout of the insulin receptor results in hyperglycemia due to impaired insulin secretion, suggesting that this cell is an important target of insulin action. The present studies were undertaken in beta-cell insulin receptor knockout (betaIRKO) mice to define the mechanisms underlying the defect in insulin secretion. On the basis of responses to intraperitoneal glucose, approximately 7-mo-old betaIRKO mice were either diabetic (25%) or normally glucose tolerant (75%). Total insulin content was profoundly reduced in pancreata of mutant mice compared with controls. Both groups also exhibited reduced beta-cell mass and islet number. However, insulin mRNA and protein were similar in islets of diabetic and normoglycemic betaIRKO mice compared with controls. Insulin secretion in response to insulin secretagogues from the isolated perfused pancreas was markedly reduced in the diabetic betaIRKOs and to a lesser degree in the nondiabetic betaIRKO group. Pancreatic islets of nondiabetic betaIRKO animals also exhibited defects in glyceraldehyde- and KCl-stimulated insulin release that were milder than in the diabetic animals. Gene expression analysis of islets revealed a modest reduction of GLUT2 and glucokinase gene expression in both the nondiabetic and diabetic mutants. Taken together, these data indicate that loss of functional receptors for insulin in beta-cells leads primarily to profound defects in postnatal beta-cell growth. In addition, altered glucose sensing may also contribute to defective insulin secretion in mutant animals that develop diabetes.

64Cu- and 68Ga-labelled [Nle(14),Lys(40)(Ahx-NODAGA)NH2]-exendin-4 for pancreatic beta cell imaging in rats.

PURPOSE Glucagon-like peptide-1 receptor (GLP-1R) is a molecular target for imaging of pancreatic beta cells. We compared the ability of [Nle(14),Lys(40)(Ahx-NODAGA-(64)Cu)NH2]-exendin-4 ([(64)Cu]NODAGA-exendin-4) and [Nle(14),Lys(40)(Ahx-NODAGA-(68)Ga)NH2]-exendin-4 ([(68)Ga]NODAGA-exendin-4) to detect native pancreatic islets in rodents. PROCEDURES The stability, lipophilicity and affinity of the radiotracers to the GLP-1R were determined in vitro. The biodistribution of the tracers was assessed using autoradiography, ex vivo biodistribution and PET imaging. Estimates for human radiation dosimetry were calculated. RESULTS We found GLP-1R-specific labelling of pancreatic islets. However, the pancreas could not be visualised in PET images. The highest uptake of the tracers was observed in the kidneys. Effective dose estimates for [(64)Cu]NODAGA-exendin-4 and [(68)Ga]NODAGA-exendin-4 were 0.144 and 0.012 mSv/MBq, respectively. CONCLUSION [(64)Cu]NODAGA-exendin-4 might be more effective for labelling islets than [(68)Ga]NODAGA-exendin-4. This is probably due to the lower specific radioactivity of [(68)Ga]NODAGA-exendin-4 compared to [(64)Cu]NODAGA-exendin-4. The radiation dose in the kidneys may limit the use of [(64)Cu]NODAGA-exendin-4 as a clinical tracer.

Polycystic Kidneys and GM2 Gangliosidosis-Like Disease in Neonatal Springboks (Antidorcas marsupialis)

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008-2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 μm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 μm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies.

Hyperplasia to neoplasia sequence of duodenal and pancreatic neuroendocrine diseases and pseudohyperplasia of the PP-cells in the pancreas.

Hyperplastic changes of the neuroendocrine cell system may have the potential to evolve into neoplastic diseases. This is particularly the case in the setting of genetically determined and hereditary neuroendocrine tumor syndromes such as MEN1. The review discusses the MEN1-associated hyperplasia-neoplasia sequence in the development of gastrinomas in the duodenum and glucagon-producing tumors in the pancreas. It also presents other newly described diseases (e.g., glucagon cell adenomatosis and insulinomatosis) in which the tumors are (or most likely) also preceded by islet cell hyperplasia. Finally, the pseudohyperplasia of PP-rich islets in the pancreatic head is defined as a physiologic condition clearly differing from other hyperplastic-neoplastic neuroendocrine diseases.

GLUCAGON GENE EXPRESSION: ANALYSIS OF PREPROGLUCAGON

Glucagon is a 29 amino acid polypeptide hormone produced in the (alpha) cells of the pancreatic islets. The purpose of this research was to understand better the role of glucagon in the regulation of metabolic processes. As with other polypeptide hormones, the synthesis of glucagon is thought to involve a larger precursor, which is then enzymatically cleaved to the functional form. The specific research objectives were to obtain cloned copies of the messenger RNA (mRNA) for pancreatic glucagon, to determine their primary sequences, and from this coding information to deduce the amino acid sequence of the initial glucagon precursor. From this suggested preproglucagon sequence and prior information on possible proglucagon intermediate processing products, the overall objective of this research is to propose a possible pathway for the biosynthesis of pancreatic glucagon.^ Synthetic oligodeoxynucleotide probes of 14-nucleotides (14-mer) and 17-nucleotides (a 17-mer) complementary to codons specifying a unique sequence of mature glucagon were synthesized. The ('32)P-labeled-14-mer was hybridized with size-fractionated fetal bovine pancreatic poly(A('+))RNA bound to nitrocellulose. RNA fractions of (TURN)14S were found to hybridize specifically, resulting in an (TURN)10-fold enrichment for these sequences. These poly(A('+))RNAs were translated in a cell-free system and the products analyzed by gel electrophoresis. The translation products were found to be enriched for a protein of the putative size of mammalian preproglucagon ((TURN)21 kd). These enriched RNA fractions were used to construct a complementary DNA (cDNA) library is plasmid pBR322.^ Screening of duplicate colony filters with the ('32)P-labeled-17-mer and a ('32)P-labeled-17-mer-primed cDNA probe indicated 25 possible glucagon clones from 3100 colonies screened. Restriction mapping of 6 of these clones suggested that they represented a single mRNA species. Primary sequence analysis of one clone containing a 1200 base pair DNA insert revealed that it contained essentially a full-length copy of glucagon cDNA.^ Analaysis of the cDNA suggested that it encoded an initial translation product of 180 amino acids with an M(,r) = 21 kd. The first initiation codon (ATG, methionine) followed by the longest open reading frame of 540 nucleotides was preceded by a 5'-untranslated region of 90 nucleotides, and was followed by a longer 3'-untranslated region of 471 nucleotides, resulting in a total of 1101 nucleotides. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Ingeniería de la conservación: Investigación aplicada para la conservación de la fauna mediterránea amenazada

El ser humano nunca ha tenido una capacidad semejante a la actual para alterar el medio en el que vive. A través fundamentalmente de cambios en el uso del hábitat, alteración de los ciclos biogeoquímicos, cambio climático y presencia de especies invasoras, la tasa de extinción de especies se ha acelerado enormemente. Al crecer más rápido la población humana dentro de las áreas en las que se concentra una mayor biodiversidad (biodiversity hotspots), las tasas de transformación son mayores, por lo que dicha biodiversidad se encuentra más amenazada. Entre los principales 25 biodiversity hotspots globales se encuentra la cuenca mediterránea. Las afecciones a la biodiversidad hacen por tanto que en dicha cuenca mediterránea sea necesario poner en práctica iniciativas innovadoras para su conservación. Pero además de una cuidadosa selección, es necesario el seguimiento y la evaluación de la eficacia y la eficienca de dichas medidas, para comprobar su idoneidad y mejorarlas cuando sea posible. Esta es la tarea de la ingeniería de la conservación. Esta Memoria busca analizar y proponer mejoras en cuatro acciones experimentales de conservación de la biodiversidad que tienen un marco común, la conservación de especies subrogadas del monte mediterráneo. En concreto de cinco especies amenazadas: conejo de monte (Oryctolagus cuniculus, especie llave), águila imperial ibérica (Aquila adalberti), águila perdicera (Aquila fasciata), cigüeña negra (Ciconia nigra) y lince ibérico (Lynx pardinus), todas ellas especies paraguas y bandera. Casi todas estas especies se enfrentan a amenazas comunes, como son la pérdida de la calidad del hábitat, la mortalidad de origen humano, la escasez de alimento o la falta de eficiencia en las técnicas de seguimiento. Las acciones destinadas a revertir o minimizar estas amenazas han tenido un marco geográfico y ecológico común, 49 fincas privadas de monte mediterráneo situadas en 9 espacios de la Red Natura 2000 del centro-occidental peninsular, en las que se ha podido trabajar merced a acuerdos de custodia del territorio. Dichas actividades han sido financiadas casi por completo con cargo a fondos LIFE Naturaleza de la Comisión Europea. El presente documento contribuye a aportar el conocimiento científico imprescindible para mejorar la eficacia y eficiencia de las medidas técnicas de gestión y conservación: la ingeniería de la conservación. Como resultados más relevantes se pueden citar que la selección del conejo de las zonas sembradas aumenta con la presencia tanto de isletas de matorral como de refugios y resulta inferior en las zonas de matorral más cerrado; que la electrocución es una de las principales causas de mortalidad de las rapaces amenazadas, así como que se ha detectado que la mortalidad se concentra en determinadas zonas que deben ser prioritarias para su corrección y que éstas deben pasar por las modificaciones estructurales; que la selección de zonas de alimentación por parte de la cigüeña negra se ve afectada por numerosos factores y que una adecuada ubicación y gestión de las charcas para ganadería y caza mayor puede favorecer no sólo la alimentación de esta especie, sino suponer un lugar de cría para diversas especies de anfibios y peces autóctonos, en franco declive; finalmente, hay que destacar que la mejora en el seguimiento de las poblaciones de lince ibérico pasa por la incorporación de métodos de seguimiento novedosos, como las cámaras infrarrojas en letrinas, dada la capacidad de aprendizaje de la especie. Abstract Nowadays the human being has reached its top ability to exchange its environment. Mainly through habitat change, altering biogeochemical cycles, climatic change and alien species, global extinction rate has increased dramatically. Population increases faster in biodiversity hotspots, thus change rates are higher. Mediterranean basin is included within the 25 most valuable biodiversity hotspots. Affections on biodiversity turns necessary to develop innovative actions to maintain the most threatened species in this hotspot. But further than a careful selection and implementarion, survey and evaluation of those practices is necessary to check its suitability. Those are actions for the conservation engineering. This Memory looks for to analyze and to put forward improvements in four experimental biodiversity conservation actions with a common framework: conservation of surrogate Mediterranean species. Those actions were specifically directed to five threatened species: the European wild rabbit (Oryctolagus cuniculus, key species), Spanish imperial eagle (Aquila adalberti) and Bonelli’s eagle (Aquila fasciata, both of the umbrella and flagship species), black stork (Ciconia nigra, umbrella and flagship species) and the Iberian lynx (Lynx pardinus, umbrella and flagship species). Those species have common threats: habitat loss, maninduced mortality, food shortening and lack of surveying efficiency. Actions aimed to minimize those threats also had a common geographical frame: 49 privately owned states within 9 Natura 2000 areas in Southwestern Spain. Actions were developed through lands tewardship agreements, and were even wholly undertaken through European Commision LIFE’s fund. This document contributes to develop cientific knowledge necessary for increasing efficacy and efficiency for implementing technical measures devoted to conservation, the conservation engineering. One of the most outstanding result might be the necessity of including bushy islets or rabbit shelter within sowings when enhacing rabbit populations is the goal. Electrocution is one of the commonest man-induced death causes for raptors, especially for the threatened ones. Areas of concentration of this mortality have been detected, and when mitigation measures have been evaluated, only structural modifications had results. The black stork’s selection of feeding areas is influenced by several factors. An adequate location and management of livestock’s ponds can favour not only this species, but also creating new breeding places for amphibians and indigenous fish species, most of them in a sharp decline. Finally, improving the survey of the Iberian lynx might be achieved through implementing newly developed surveying systems, such as the infrared cameras in latrines.

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8+ T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8+ T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8+ T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8+ T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8+ T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8+ T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

Expression of adenoviral E3 transgenes in β cells prevents autoimmune diabetes

The adenovirus (Ad) genome contains immunoregulatory and cytokine inhibitory genes that are presumed to function in facilitating acute infection or in establishing persistence in vivo. Some of these genes are clustered in early region 3 (E3), which contains a 19-kDa glycoprotein (gp19) that inhibits the transport of selected class I major histocompatibility complex (MHC) molecules out of the endoplasmic reticulum. In addition, the E3 region contains three protein inhibitors of the cytolytic function of tumor necrosis factor α (TNF-α). Because type I autoimmune diabetes destroys islets by mechanisms that involve class I MHC and TNF-α, we investigated whether the entire cassette of Ad E3 genes might prevent the onset of diabetes in a well studied lymphocytic choriomeningitis viral (LCMV) murine model of virus-induced autoimmune diabetes. In this model, a LCMV polypeptide (either glycoprotein or nucleoprotein) expressed as a transgene in the islets is a target for autoimmune destruction of β cells after LCMV infection. In this scenario the LCMV-induced immune response is directed not only against the virus but also against the LCMV transgenes expressed in the β cells. Our experiments demonstrated a very efficient prevention of this LCMV-triggered diabetes by the Ad E3 genes. This resulted from the inhibition of target cell recognition by a fully competent and LCMV-primed immune system. Unlike the results from the β-2 microglobulin gene deletion experiments, our approach shows that selective regulation at the level of the target cell is sufficient to prevent autoimmune diabetes without disrupting the function of the systemic immune response. Although the Ad genes in these experiments were provided as transgenes, recent experiments may permit the introduction of such genes through the use of viral vectors. Although the decrease in class I MHC in islets by Ad genes was demonstrated in these in vivo studies, the relative importance of this process and the control of TNF-α cytolysis must await further genetic dissection of the introduced Ad genes.

Pancreas development is promoted by cyclopamine, a Hedgehog signaling inhibitor

Exposure to cyclopamine, a steroid alkaloid that blocks Sonic hedgehog (Shh) signaling, promotes pancreatic expansion in embryonic chicks. Heterotopic development of pancreatic endocrine and exocrine structures occurs in regions adjacent to the pancreas including stomach and duodenum, and insulin-producing islets in the pancreas are enlarged. The homeodomain transcription factor PDX1, required for pancreas development, is expressed broadly in the posterior foregut but pancreas development normally initiates only in a restricted region of PDX1-expressing posterior foregut where endodermal Shh expression is repressed. The results suggests that cyclopamine expands the endodermal region where Shh signaling does not occur, resulting in pancreatic differentiation in a larger region of PDX1-expressing foregut endoderm. Cyclopamine reveals the capacity of a broad region of the posterior embryonic foregut to form pancreatic cells and provides a means for expanding embryonic pancreas development.

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca2+ signaling, and insulin secretion in the anx7(+/−) knockout mouse

The mammalian anx7 gene codes for a Ca2+-activated GTPase, which supports Ca2+/GTP-dependent secretion events and Ca2+ channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca2+ signaling in secreting pancreatic β cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the β cells. The nullizygous anx7 (−/−) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/−) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/−) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca2+ channel functions are normal. However, electrooptical recordings indicate that the (+/−) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP3)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP3 receptor expression and function in pancreatic islets. The profound increase in islets, β cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic β cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca2+ signaling through IP3-sensitive Ca2+ stores.

The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.