Insulin, synthesized by the beta cells of pancreatic islets, is of major physiological importance in metabolic homeostasis. While mature insulin consists of two polypeptide chains joined by disulphide bridges, the gene encodes for a highly conserved single chain precursor, preproinsulin [1]. In most species preproinsulin exists as a single gene, whereas in the mouse and the rat two non-allelic insulin genes are present. The human insulin gene is located on the short arm of chromosome 11 (p15.5) [2], the rat insulin I and II genes are colocalized on chromosome 1 [3] and the mouse genes are positioned on two different chromosomes, insulin I on chromosome 19 [4] and insulin II on chromosome 7 [5]. In adult islets, the nonallelic genes appear to be coordinately expressed and regulated [6, 7]. The rodent insulin II and the human genes contain three exons and two introns, whilst insulin I lacks the second intron. The organisation and structure of the insulin gene has been reviewed in detail [8]. Insulin is regulat- AbstractThe mammalian insulin gene is exclusively expressed in the beta cells of the endocrine pancreas. Two decades of intensive physiological and biochemical studies have led to the identification of regulatory sequence motifs along the insulin promoter and to the isolation of transcription factors which interact to activate gene transcription. The majority of the islet-restricted (BETA2, PDX-1, RIP3b1-Act/C1) and ubiquitous (E2A, HEB) insulin-binding proteins have been characterized. Transcriptional regulation results not only from specific combinations of these activators through DNA-protein and protein-protein interactions, but also from their relative nuclear concentrations, generating a cooperativity and transcriptional synergism unique to the insulin gene. Their DNA binding activity and their transactivating potency can be modified in response to nutrients (glucose, NEFA) or hormonal stimuli (insulin, leptin, glucagon like peptide-1, growth hormone, prolactin) through kinase-dependent signalling pathways (PI3-K, p38MAPK, PKA, CaMK) modulating their affinities for DNA and/or for each other. From the overview of the research presented, it is clear that much more study is required to fully comprehend the mechanisms involved in the regulated-expression of the insulin gene in the beta cell to prevent its impairment in diabetes. [Diabetologia (2002) 45: 309±326]

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR ‫؍‬ 1.29, P ‫؍‬ 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.insulin ͉ polymorphisms ͉ TGF-␤ ͉ type 2 diabetes C omponents of both the exocrine and endocrine pancreas are affected by diseases, e.g., pancreatic cancer and type 2 diabetes mellitus (T2DM), which severely compromise both the quality and span of human life. Both glandular compartments share the same cellular origin and early morphogenetic pathways, suggesting a close functional and pathophysiological relationship. For instance, the exocrine-specific transcription factor p48 and the endocrine-specific pancreatic duodenal homeobox gene 1 (PDX-1) are both expressed in the common cell precursor (1); and, under pathological conditions their compartmentalization may be lost, as exemplified by the detection of PDX-1 in pancreatic cancer (2). In fact, T2DM is both a common feature and a risk factor for the subsequent development of pancreatic cancer (3, 4). The TGF-␤-inducible transcription factor KLF11 regulates exocrine cell growth and behaves as a tumor suppressor in pancreatic cancer (ref. 5 and M.E.F.-Z. and R.U., unpublished observation). Because the TGF-␤ signaling pathway is also a major regulator of endocrine cell fate (1, 6), the current study has been designed to define the role of the Sp1-like transcription factor KLF11 in the biology of ...

Type 1 diabetes is characterized by the infiltration of inflammatory cells into pancreatic islets of Langerhans, followed by the selective and progressive destruction of insulin-secreting beta cells. Isletinfiltrating leukocytes secrete cytokines such as IL-1␤ and IFN-␥, which contribute to beta cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-B is an important component of the signal triggering beta cell apoptosis. To study the in vivo role of NF-B in beta cell death, we generated a transgenic mouse line expressing a degradation-resistant NF-B protein inhibitor (⌬NI B␣), acting specifically in beta cells, in an inducible and reversible manner, by using the tet-on regulation system. In vitro, islets expressing the ⌬NI B␣ protein were resistant to the deleterious effects of IL-1␤ and IFN-␥, as assessed by reduced NO production and beta-cell apoptosis. This effect was even more striking in vivo, where nearly complete protection against multiple low-dose streptozocin-induced diabetes was observed, with reduced intraislet lymphocytic infiltration. Our results show in vivo that beta cell-specific activation of NF-B is a key event in the progressive loss of beta cells in diabetes. Inhibition of this process could be a potential effective strategy for beta-cell protection.apoptosis ͉ cytokine ͉ diabetes ͉ transgenic mice ͉ insulin

In type 2 diabetes, chronic hyperglycemia has been suggested to be detrimental to beta-cell function, causing reduced glucose-stimulated insulin secretion and disproportionately elevated proinsulin. In the present study, we investigated the effect on several beta-cell functions of prolonged in vitro exposure of human pancreatic islet cultures to high glucose concentrations. Islets exposed to high glucose levels (33 mmol/l) for 4 and 9 days showed dramatic decreases in glucose-induced insulin release and in islet insulin content, with increased proportion of proinsulin-like peptides relative to insulin. The depletion in insulin stores correlated with the reduction in insulin mRNA levels and human insulin promoter transcriptional activity. We also demonstrated that high glucose dramatically lowered the binding activity of pancreatic duodenal homeobox 1 (the glucose-sensitive transcription factor), whereas the transcription factor rat insulin promoter element 3b1 activator was less influenced and insulin enhancer factor 1 remained unaffected. Most of these beta-cell impairments were partially reversible when islets first incubated for 6 days in high glucose were transferred to normal glucose (5.5 mmol/l) concentrations for 3 days. We conclude that cultured human islets are sensitive to the deleterious effect of high glucose concentrations at multiple functional levels, and that such mechanisms may play an important role in the decreased insulin production and secretion of type 2 diabetic patients.

In cultured rat and human pancreatic islets, glucose stimulated transcription of the rat insulin I gene through the minienhancer (FF) located between residues -196 and -247. The glucose-sensitive element was delineated to the region -193 to -227. The minienhancer bound islet nuclear proteins to form three major complexes (C1-C3). A 22-bp subfragment, spanning the sequence -206 to -227, was sufficient to retain all binding activities of the entire FF. The homologous sequence ofthe human insulin promoter interacted with rat islet nuclear extracts to form a single complex, corresponding to the Cl complex of the rat insulin I sequence.Cl was present only in insulin-producing cells; it was the major complex detected in isolated human islets with both rat and human insulin sequences. Furthermore, the DNA binding activity of the C1 factor(s) was selectively modulated by extracellular glucose in a dose-dependent manner; a 4.5-fold increase in binding intensity was detected when rat islets were incubated for 1-3 h in the presence of 20 vs. 1-2 mM glucose. We therefore suggest that the factor(s) involved in the Cl complex corresponds to the glucose-sensitive factor and, consequently, may play a determining role in glucose-regulated expression of the insulin gene.Insulin gene expression is restricted to pancreatic beta cells. It has been shown by transfection of cultured cells (1, 2) and by gene transfer in mouse embryos (3) that the regulation occurs at the level of transcription and is controlled by cis-acting elements in the 5' flanking region of the gene (4-10). These elements bind multiple beta-cell proteins (9)(10)(11)(12)(13)(14)(15)(16)(17)(18), providing the basis for identifying and cloning putative insulin gene transcription factors (19)(20)(21)(22).Insulin production is regulated by glucose (23-25). The sugar acts both by increasing mRNA stability and by stimulating the transcription rate of the insulin gene (26)(27)(28)(29)(30). Recently, the expression of a chimeric gene containing the rat insulin I minienhancer was reported to be stimulated by glucose in transfected rat fetal islets (31). Fetal beta cells fail to recognize nutrients as inducers of the normal stimulussecretion coupling (32,33). It is therefore important to establish that in normal adult pancreatic islets, where glucose is the main physiologic stimulator, insulin synthesis is similarly activated through the interaction of trans-acting transcription factors with a cis-acting regulatory element of the insulin enhancer.In this study, we describe an insulin-specific DNA binding protein whose binding activity is sensitive to extracellular glucose, providing evidence of a glucose-sensitive islet nuclear protein that may play a crucial role in glucose-regulated expression of the insulin gene. MATERIALS AND METHODSIslet Isolation and Transfection. Male adult SpragueDawley or Sabra rats (Hebrew University) were used. Pancreatic islets were isolated by a modification ofthe method of Lacy and Kostianovsky (34); 200-300 rat islets and "100 human i...

Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.

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.

The PDX-1 transcription factor plays a key role in pancreatic development and in the regulation of the insulin gene in the adult ␤ cell. As its functions appear to be similar in humans and mice, we analyzed the functional conservation of homologous sequences important for the maintenance and the cell-specific regulation of the pdx-1 gene. Apart from the proximal promoter region, three highly homologous (PH1 to PH3) sequences were apparent in the human and mouse 5 flanking regions of the gene. By transient transfections in ␤ and non-␤ cells, we show that mainly PH1 and PH2 preferentially confer ␤-cell-specific activation on a heterologous promoter. DNase I footprinting and binding analyses revealed that both bind to and are transactivated by hepatocyte nuclear factor 3␤ (HNF-3␤). Furthermore, the PH1 enhancer element also binds the PDX-1 transcription factor itself, which acts cooperatively with adjacent HNF-3␤ to regulate its transcriptional potency. This finding suggests a possible autoregulatory loop as a mechanism for PDX-1 to control its own expression.The mammalian pancreas develops from the endoderm in the upper duodenal part of the foregut as dorsal and ventral buds which fuse together to form the organ. Recently, considerable progress has been made in identifying genes that control the embryonic development of the islet. Most are transcription factors such as isl-1, PDX-1, BETA2 (also called NeuroD), Nkx 2.2, Pax4, and Pax6 (for reviews, see references 8, 9, 14, 15, 26, 30, and 31). The first molecular marker identified that specifies the early pancreatic epithelium was the homeodomain-containing transcription factor PDX-1 (1, 2, 13). PDX-1 function appears to be well conserved. Its absence in both humans and mice leads to agenesis of the pancreas, while in the adult pancreatic islet, its expression is restricted to the ␤ cell, where it acts as the glucose-sensitive transcription factor of the insulin gene (19).Initiation of transcription appears to be the major level of regulation for many cell type-specific genes. To elucidate the regulation of pdx-1 gene expression, a 6.5-kb fragment upstream of the transcription start site of the rat pdx-1 gene (also called stf-1) (28) and a fragment extending from the region from kb Ϫ4.5 to ϩ8.2 of the mouse pdx-1 gene (33) were shown to direct the expression of the ␤-galactosidase (␤-Gal) reporter gene to pancreatic islet cells in transgenic mice. Using transiently transfected ␤ cells, it was shown that the expression of the rat pdx-1 gene, apart from a proximal E box, depended on a distal enhancer element that was activated by the cooperative action of the endodermal transcription factors hepatocyte nuclear factor 3␤ (HNF-3␤) and BETA2 (27). A study of the mouse pdx-1 promoter revealed that the region involved in regulating ␤-cell-specific transcription and directing appropriate developmental and adult-specific expression in transgenic animals contained an HNF-3-like element (33). Thus, these initial studies with the rat and the mouse enhancer-promoter regions su...