Mehboob A. Hussain

Glucagon Gene Transcription Activation Mediated by Synergistic Interactions of pax-6 and cdx-2 with the p300 Co-activator

In the endocrine pancreas, α-cell-specific expression of the glucagon gene is mediated by DNA-binding proteins that interact with the proximal G1 promoter element, which contains several AT-rich domains. The homeodomain transcription factors brain-4, pax-6, and cdx-2 have been shown to bind to these sites and to transactivate glucagon gene expression. In the present study, we investigated the interaction of cdx-2 and pax-6 with p300, a co-activator coupled to the basal transcription machinery. In transient transfection-expression experiments, we found that the transactivating effects of cdx-2 and pax-6 on the glucagon gene were greatly enhanced by the additional expression of p300. This enhancement was due to direct protein-protein interactions of both pax-6 and cdx-2 with the N-terminal C/H1 domain of p300. pax-6 and cdx-2 also directly interacted with one another at the protein level. pax-6, bound to its DNA recognition site in the glucagon G1 promoter element, tethered cdx-2 to the molecular complex of pax-6 and p300. Further, we found that the presence of cdx-2 enhanced the interaction of pax-6 with p300, thus establishing a molecular complex of transcription factors implicated in tissue-specific glucagon gene expression with the basal transcriptional machinery.

POU domain transcription factor brain 4 confers pancreatic alpha-cell-specific expression of the proglucagon gene through interaction with a novel proximal promoter G1 element.

The proglucagon gene is expressed in a highly restricted tissue-specific manner in the alpha cells of the pancreatic islet, the hypothalamus, and the small and large intestines. Proglucagon is processed to glucagon and glucagon-like peptides GLP-1 and -2. Glucagon is expressed in alpha cells and regulates glucose homeostasis. GLP-1 is implicated in the control of insulin secretion, food intake, and satiety signaling, and GLP-2 is implicated in regulating small-bowel growth. Cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element which contains several AT-rich domains with binding sites for homeodomain transcription factors. In an attempt to identify major homeodomain proteins involved in pancreatic alpha-cell-specific proglucagon expression, we found that the POU domain transcription factor brain 4 is abundantly expressed in proglucagon-producing islet cell lines and rat pancreatic islets. In the latter, brain 4 and glucagon immunoreactivity colocalize in the outer mantle of islets. Electrophoretic mobility shift assays with specific antisera identify brain 4 as a major constituent of nuclear proteins of glucagon-producing cells that bind to the G1 element of the proglucagon gene proximal promoter. Transcriptional transactivation experiments reveal that brain 4 is a major regulator of proglucagon gene expression by its interaction with the G1 element. The finding that a neuronal transcription factor is involved in glucagon gene transcription may explain the presence of proglucagon in certain areas of the brain as well as in pancreatic alpha cells. Further, this finding supports the idea that the neuronal properties of endodermis-derived endocrine pancreatic cells may find their basis in regulation of gene expression by neuronal transcription factors.

Increased Pancreatic β-Cell Proliferation Mediated by CREB Binding Protein Gene Activation

ABSTRACT The cyclic AMP (cAMP) signaling pathway is central in β-cell gene expression and function. In the nucleus, protein kinase A (PKA) phosphorylates CREB, resulting in recruitment of the transcriptional coactivators p300 and CREB binding protein (CBP). CBP, but not p300, is phosphorylated at serine 436 in response to insulin action. CBP phosphorylation disrupts CREB-CBP interaction and thus reduces nuclear cAMP action. To elucidate the importance of the cAMP-PKA-CREB-CBP pathway in pancreatic β cells specifically at the nuclear level, we have examined mutant mice lacking the insulin-dependent phosphorylation site of CBP. In these mice, the CREB-CBP interaction is enhanced in both the absence and presence of cAMP stimulation. We found that islet and β-cell masses were increased twofold, while pancreas weights were not different from the weights of wild-type littermates. β-Cell proliferation was increased both in vivo and in vitro in isolated islet cultures. Surprisingly, glucose-stimulated insulin secretion from perfused, isolated mutant islets was reduced. However, β-cell depolarization with KCl induced similar levels of insulin release from mutant and wild-type islets, indicating normal insulin synthesis and storage. In addition, transcripts of pgc1a, which disrupts glucose-stimulated insulin secretion, were also markedly elevated. In conclusion, sustained activation of CBP-responsive genes results in increased β-cell proliferation. In these β cells, however, glucose-stimulated insulin secretion was diminished, resulting from concomitant CREB-CBP-mediated pgc1a gene activation.

Exendin-4 Stimulation of Cyclin A2 in β-Cell Proliferation

OBJECTIVE—β-Cell proliferation is an important mechanism underlying β-cell mass adaptation to metabolic demands. We have examined effects, in particular those mediated through intracellular cAMP signaling, of the incretin hormone analog exendin-4 on cell cycle regulation in β-cells. RESEARCH DESIGN AND METHODS—Changes in islet protein levels of cyclins and of two critical cell cycle regulators cyclin kinase inhibitor p27 and S-phase kinase–associated protein 2 (Skp2) were assessed in mice treated with exendin-4 and in a mouse model with specific upregulation of nuclear cAMP signaling exhibiting increased β-cell proliferation (CBP-S436A mouse). Because cyclin A2 was stimulated by cAMP, we assessed the role of cylcin A2 in cell cycle progression in Min6 and isolated islet β-cells. RESULTS—Mice treated with exendin-4 showed increased β-cell proliferation, elevated islet protein levels of cyclin A2 with unchanged D-type cyclins, elevated PDX-1 and Skp2 levels, and reduced p27 levels. Exendin-4 stimulated cyclin A2 promoter activity via the cAMP–cAMP response element binding protein pathway. CBP-S436A islets exhibited elevated cyclin A2, reduced p27, and no changes in D-type cyclins, PDX-1, or Skp2. In cultured islets, exendin-4 increased cyclin A2 and Skp2 and reduced p27. Cyclin A2 overexpression in primary islets increased proliferation and reduced p27. In Min6 cells, cyclin A2 knockdown prevented exendin-4–stimulated proliferation. PDX-1 knockdown reduced exendin-4–stimulated cAMP synthesis and cyclin A2 transcription. CONCLUSIONS—Cyclin A2 is required for β-cell proliferation, exendin-4 stimulates cyclin A2 expression via the cAMP pathway, and exendin-4 stimulation of cAMP requires PDX-1.

Beta-cell apoptosis in the pathogenesis of human type 2 diabetes mellitus.

Type 2 diabetes is accompanied by chronic insulin resistance and a progressive decline in b-cell function (1). Obesity is a major risk factor for the development of type 2 diabetes (2, 3) and is thought to confer increased risk for type 2 diabetes through obesity-associated insulin resistance (4). However, most people who are obese do not develop diabetes but compensate their relative insulin resistance by increasing insulin secretion (5). In rodent models of obesity without diabetes there is (as opposed to non-obese littermates) an adaptive increase in b-cell mass to meet metabolic demands (6). Although not many data are available, studies suggest that b-cell mass is also adaptively increased in non-diabetic obese humans (7, 8). b-cell mass is regulated by a balance of b-cell replication and apoptosis, as well as development of new islets from exocrine pancreatic ducts (neogenesis) (9, 10). Disruption of any of the pathways of b-cell formation or increased rates of b-cell death would result in decreased b-cell mass and thus reduced capacity to produce insulin. There is controversy whether b-cell mass is decreased in type 2 diabetes mellitus (8, 11–17). These discrepancies are in part due to the paucity of available data in humans. Furthermore, it is controversial whether b-cell apoptosis is truly increased in type 2 diabetes. A recent (18) very carefully conducted study gives new and convincing data indicating that increased apoptosis rather than decreased neogenesis or replication may be the main mechanism leading to reduced b-cell mass in type 2 diabetics. Autopsy material from obese patients with diabetes or with impaired fasting glucose (IFG) or without diabetes as well as from lean patients was examined. The authors report that obesity in non-diabetic humans is accompanied by a 50% increase in relative b-cell volume as compared with lean non-diabetic humans. However, the non-diabetic obese humans died younger than the non-diabetic lean humans and the difference found on autopsy may be due to difference in age. Obese humans with IFG and type 2 diabetes had a respective 40 and 63% deficit in relative b-cell volume. The decreased b-cell volume in patients with type 2 diabetes was due to a reduced number of b-cells rather than a smaller volume of individual cells and occurred irrespectively of whether they were treated with diet alone or oral medications or insulin. There was no difference in the mean fasting plasma glucose levels among these three treatment groups. Lean subjects with type 2 diabetes had a 41% deficit in relative b-cell volume compared with lean non-diabetic subjects. These findings are consistent with other carefully conducted recent studies in which b-cell mass is decreased in type 2 diabetes (8, 15, 17). Neogenesis, while increased in obesity, was comparable in all groups. Decreased b-cell replication was found with aging (18). Because new islet formation, the predominant input into the b-cell mass, appears intact in type 2 diabetics (18), the mechanism for the decreased b-cell mass would have to be increased b-cell apoptosis. Indeed, a significantly increased frequency of apoptotic events were detected in lean type 2 diabetic vs non-diabetic cases. When normalized to the b-cell volume, the frequency of apoptosis was 3-fold higher in obese cases of type 2 diabetes and 10-fold higher in lean cases of type 2 diabetics as compared with their controls. Thus, relative b-cell volume and therefore b-cell mass is decreased in both obese and lean humans with type 2 diabetes compared with non-diabetic ageand weight-matched controls. The fact that patients with IFG, a risk group for developing diabetes, had a 40% deficit in relative b-cell volume indicates that loss of b-cells is an early process in the pathogenesis of diabetes mellitus (18). Once b-cell mass decreases below a critical level and insulin production no longer meets metabolic demands we have hyperglycemia. Does a 60% decrease in b-cell mass – as found in this study – translate into impaired glucose metabolism? Humans who have undergone 50% pancreatectomy have impaired glucose tolerance and insulin secretion in response to a hyperglycemic clamp (19 –23). On this basis we may assume that a 60% reduction in b-cell mass in the face of insulin resistance may be sufficient to result in hyperglycemia. What are the mechanisms for the increased apoptosis found in the islets of type 2 diabetics? The islet in type 2 diabetes is characterized by deposits of polypeptide (IAPP) (15, 24–29). This peptide causes apoptosis of b-cells (30, 31), particularly when it is in H IG H L IG H T European Journal of Endocrinology (2003) 149 99–102 ISSN 0804-4643

Misexpression of the pancreatic homeodomain protein IDX-1 by the Hoxa-4 promoter associated with agenesis of the cecum

BACKGROUND & AIMS The endoderm-specific homeodomain transcription factor IDX-1 is critical for pancreas development and for the regulation of islet cell-specific genes. During development, IDX-1 is expressed in the epithelial cells of the endoderm in the pancreatic anlage of the foregut. The aim of this study was to determine whether IDX-1 may have potential properties of a master homeotic determinant of pancreas and/or gut development. METHODS Transgenic mice were generated in which the expression of IDX-1 was misdirected by a promoter of the mesoderm-specific homeodomain protein Hoxa-4 known to express in the stomach and hindgut during development. The expectation was the formation of ectopic pancreatic tissue or alterations of gut patterning or morphology. RESULTS Although no ectopic induction of pancreatic markers was found in these transgenic mice, they manifested an altered midgut-hindgut union and agenesis of the cecum. Further, IDX-1 binds to the gut-specific homeodomain protein Cdx-2 and inhibits transactivation of the sucrase-isomaltase promoter by Cdx-2. CONCLUSIONS These findings further support the emerging understanding that interactions among different classes of homeodomain proteins, expressed in a spatially and temporally restricted manner during development, determine the pattern of organogenesis. A possible mechanism for the dysmorphogenesis of the proximal colon may be an inhibition of Cdx-2 actions by IDX-1.

Molecular basis of pancreas development and function

1. An Historical and Phylogenetic Perspective of Islet-Cell Development R.C. Heller, J. Jensen, O.D. Madsen, H.V. Petersen, P. Serup. 2. Glucose Signalling to Transcription Factors of the Insulin Gene D. Melloul. 3. beta-Cell Dysfunction and Chronic Hyperglycaemia J.L. Leahy. 4. Glucose Toxicity of the Pancreatic beta-cell C. Gleason, J. Harmon, V. Poitout, R.P. Robertson, G. Sacchi, Y. Tanaka, Phuong Oanh T. Tran. 5. The alpha-Cell and Regulation of Glucagon Gene Transcription W. Knepel. 6. The KATP Channel and the Sulfonylurea Receptor T. Miki, S. Seino, H. Yano. 7. Glucagon-Like Peptide-1: An Insulinotropic Hormone with Potent Growth Factor Actions at the Pancreatic Islets of Langerhans G.G. Holz, C.A. Leech. 8. Communication of Islet Cells: Molecules and Functions D. Bosco, P. Meda. 9. Soluble Factors Important for Pancreas Development P. Czernichow, R. Scharfmann. 10. Role of Mesenchymal-Epithelial Interactions in Pancreas Development C.A. Crisera, G.K. Gittes, A.S. Kadison, M.T. Longaker, T.S. Maldonado. 11. Homeodomain Proteins in Pancreas Development U. Ahlgren, H. Edlund. 12. bHLH Factors and Notch in Pancreatic Development L.E. Flores, R.S. Heller, J. Jensen, O.D. Madsen, P. Serup. 13. Identification, Biological Functions, and Contribution to Human Diabetes of Islet-Brain 1 C.Bonny, G. Waeber. 14. Pax4 and Pax6 in Islet Differentiation P. Gruss, Xunlei Zhou. 15. The Role of the Hepatocyte Nuclear Factor Network in Glucose Homeostasis M. Stoffel. 16. Regulation of PDX-1 Gene Expression M.A. Cissell, K. Gerrish, S. Samaras, R. Stein, C.V.E. Wright. 17. Mechanisms of Postnatal b-Cell Mass Regulation S. Bonner-Weir, G.C. Weir. 18. Genetic Models of Insulin Resistance: Alterations in b-Cell Biology C.R. Kahn, R.N. Kulkarni. 19. Clinical Consequences of Genetic Defects in b-Cell Genes J.C. Evans, T.M. Frayling, A.T. Hattersley. 20. Pathophysiology of Glut2 in Diabetes Mellitus B. Thorens. 21. Use of a Cre-LoxP Strategy in Mice to Determine the Cell-Specific Roles of Glucokinase in MODY-2 M.A. Magnuson, C. Postic. 22. Development of b-Cell Lines for Transplantation in Type 1 Diabetes Mellitus S. Efrat. 23. Gene Therapeutic Approaches for b-Cell Replacement G.M. Beattie, A. Hayek, F. Levine. Colour Figures. Index.