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D Melloul

Publications and source records attributed to D Melloul.

At least 19 recordsLinked to original sources
PubMed2006

Conditional and specific NF-kappaB blockade protects pancreatic beta cells from diabetogenic agents.

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. Islet-infiltrating leukocytes secrete cytokines such as IL-1beta and IFN-gamma, which contribute to beta cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-kappaB is an important component of the signal triggering beta cell apoptosis. To study the in vivo role of NF-kappaB in beta cell death, we generated a transgenic mouse line expressing a degradation-resistant NF-kappaB protein inhibitor (DeltaNIkappaBalpha), acting specifically in beta cells, in an inducible and reversible manner, by using the tet-on regulation system. In vitro, islets expressing the DeltaNIkappaBalpha protein were resistant to the deleterious effects of IL-1beta and IFN-gamma, 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-kappaB 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.

Active Transport, Cell Nucleus↗
PubMed2002

Regulation of insulin gene transcription.

The 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.

Animals↗
PubMed2001

A pancreatic beta -cell-specific enhancer in the human PDX-1 gene is regulated by hepatocyte nuclear factor 3beta (HNF-3beta ), HNF-1alpha, and SPs transcription factors.

The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the beta-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in beta-cells, we constructed a series of 5' and 3' deletion fragments of the 5'-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in beta- and non-beta-cells a novel beta-cell-specific distal enhancer element located between -3.7 and -3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3beta (HNF-3beta) and HNF-1alpha. Cotransfection experiments suggest that HNF-3beta, HNF-1alpha, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors.

3T3 Cells↗
PubMed2001

beta-cell glucotoxicity in the Psammomys obesus model of type 2 diabetes.

Deficient insulin secretion and relative hyperproinsulinemia are characteristic features of type 2 diabetes. The gerbil Psammomys obesus appears to be an ideal natural model of the human disease because it shows increased tendency to develop diet-induced diabetes, which is associated with moderate obesity. The disease is characterized by initial hyperinsulinemia, progressing to hypoinsulinemia associated with depleted pancreatic insulin stores and an increased proportion of insulin precursor molecules in the blood and islets. Although the proinsulin translational efficacy was found to be increased in hyperglycemic animals, insulin mRNA levels were not augmented and exhibited a gradual decrease with disease progression. The development of hyperglycemia was associated with a transient increase in beta-cell proliferative activity, as opposed to a prolonged increase in the rate of beta-cell death, culminating in disruption of islet architecture. The hypothesis that glucotoxicity is responsible in part for these in vivo changes was investigated in vitro in primary islet cultures. Islets from diabetes-prone P. obesus cultured at high glucose concentrations displayed changes in beta-cell function that mimic those observed in diabetic animals. These changes include deficient insulin secretion, depleted insulin content, an increased proportion of insulin precursor molecules, a progressive increase of DNA fragmentation, and a transient proliferative response. Furthermore, insulin mRNA was not increased by short-term exposure of P. obesus islets to elevated glucose in vitro. It is proposed that beta-cell glucotoxicity in P. obesus results from the inability of proinsulin biosynthesis to keep pace with chronic insulin hypersecretion. The resulting depletion of the insulin stores may be related to deficient glucose-regulated insulin gene transcription, possibly due to defective PDX-1 (pancreatic duodenal homeobox factor-1) expression in the adult P. obesus. An additional glucotoxic effect involves the loss of beta-cell mass in hyperglycemic P. obesus as a result of progressive beta-cell death without an adequate increase in the rate of beta-cell proliferation.

Animals↗
PubMed2001

Regulatory elements involved in human pdx-1 gene expression.

PDX-1 was shown to be expressed early during development in cells of both exocrine and endocrine origin; later it becomes restricted primarily to beta-cells where it regulates the expression of beta-cell-specific genes and mediates the glucose effect on insulin gene transcription. Therefore, it was important to identify the molecular mechanisms that specifically govern the expression of pdx-1 in the mature beta-cell. To address this question, we analyzed 7 kb of the 5' flanking region of the human pdx-1 gene. By transient transfections of beta- and non-beta-cell lines with different 5' and 3' deletions of that region, a strong beta-cell-specific enhancer element located between -3.71 and 3.46 kb was revealed. We also sequenced about 4.5 kb of the human 5' flanking region and compared it with that of the mouse pdx-1 gene. This comparison revealed three short conserved regions, designated PH1, PH2, and PH3. We showed that HNF-3beta can bind and stimulate the activity of the human PH1 and PH2 elements in non-beta-cells. Results reported by Wu et al. (7) and Sharma et al. (6) also indicate that expression of the mouse pdx-1 is controlled by an HNF-3-like element. Thus, it can be stated that at least some aspects of pdx-1 expression rely on the transcription factor HNF-3beta. Because HNF-3beta is not restricted to beta-cells, the selective transcription of pdx-1 is likely to rely on additional factors. Our findings that the PH1 enhancer element binds both HNF-3beta and PDX-1 and that mutations in each individual site dramatically impair its transcriptional activity suggest that these factors cooperate with one another. We therefore propose that a possible feedback mechanism might control the expression of pdx-1 at different stages during development.

Animals↗
PubMed2001

IPF1/PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal With type 2 diabetes.

The homeodomain transcription factor IPF1/PDX1 is required in beta-cells for efficient expression of insulin, glucose transporter 2, and prohormone convertases 1/3 and 2. Psammomys obesus, a model of diet-responsive type 2 diabetes, shows markedly depleted insulin stores when given a high-energy (HE) diet. Despite hyperglycemia, insulin mRNA levels initially remained unchanged and then decreased gradually to 15% of the basal level by 3 weeks. Moreover, insulin gene expression was not increased when isolated P. obesus islets were exposed to elevated glucose concentrations. Consistent with these observations, no functional Ipf1/Pdx1 gene product was detected in islets of newborn or adult P. obesus using immunostaining, Western blot, DNA binding, and reverse transcriptase-polymerase chain reaction analyses. Other beta-cell transcription factors (e.g., ISL-1, Nkx2.2, and Nkx6.1) were expressed in P. obesus islets, and the DNA binding activity of the insulin transcription factors RIPE3b1-Act and IEF1 was intact. Ipf1/Pdx1 gene transfer to isolated P. obesus islets normalized the defect in glucose-stimulated insulin gene expression and prevented the rapid depletion of insulin content after exposure to high glucose. Taken together, these results suggest that the inability of P. obesus islets to adapt to dietary overload, with depletion of insulin content as a consequence, results from IPF1/PDX1 deficiency. However, because not all animals become hyperglycemic on HE diet, additional factors may be important for the development of diabetes in this animal model.

Adenoviridae↗
PubMed2000

Functional conservation of regulatory elements in the pdx-1 gene: PDX-1 and hepatocyte nuclear factor 3beta transcription factors mediate beta-cell-specific expression.

The PDX-1 transcription factor plays a key role in pancreatic development and in the regulation of the insulin gene in the adult beta 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 beta and non-beta cells, we show that mainly PH1 and PH2 preferentially confer beta-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 3beta (HNF-3beta). Furthermore, the PH1 enhancer element also binds the PDX-1 transcription factor itself, which acts cooperatively with adjacent HNF-3beta to regulate its transcriptional potency. This finding suggests a possible autoregulatory loop as a mechanism for PDX-1 to control its own expression.

Animals↗
PubMed1999

Regulation of the insulin gene by glucose: stimulation of trans-activation potency of human PDX-1 N-terminal domain.

The beta cells in pancreatic islets of Langerhans increase insulin gene transcription in response to glucose. The pancreatic and duodenal homeobox-1 (PDX-1) plays a major role in glucose-induced insulin transcription. We studied the functional regions of the human PDX-1 protein fused to the DNA-binding domain of the transcription factor Gal4. The results indicate that the N-terminal domain of the hPDX-1, required for transactivation (amino acids 1-120) in transfected betaTC6 and HeLa cells, is also regulated by extracellular glucose concentrations in transfected rat islets. Deletion analyses have led to the mapping of two regions within the N terminus that are essential for its trans-activation properties. One sequence spans amino acids 97-120 in transfected islet and HeLa cells or amino acids 77-120 in betaTC6 cells; the other includes the highly conserved B box (amino acids 31-41). We thus present evidence of a glucose effect on hPDX-1 trans-activation activity, in addition to the previously described regulatory effect on its DNA-binding activity.

Amino Acid Sequence↗
PubMed1999

Impaired beta-cell functions induced by chronic exposure of cultured human pancreatic islets to high glucose.

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.

Adult↗
PubMed1996

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

The beta 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 beta cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated beta 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 alpha TC1 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 beta TC1 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 beta cells.

Amino Acid Sequence↗
PubMed1994

Progressive de novo DNA methylation at the bcr-abl locus in the course of chronic myelogenous leukemia.

De novo methylation of CpG islands is a rare event in mammalian cells. It has been observed in the course of developmental processes, such as X chromosome inactivation and genomic imprinting. The methylation of DNA, an important factor in the epigenetic control of gene expression, may also be involved in tumorigenesis. After the t(9;22) chromosomal translocation and generation of the Philadelphia chromosome, the initiating event in chronic myelogenous leukemia (CML), most of the abl coding sequence is fused to the 5' region of the bcr gene. Expression of the hybrid bcr-abl gene is, therefore, regulated by the bcr promoter. In most cases of CML, one of the two abl promoters (Pa) is nested within the bcr-abl transcriptional unit and should be able to transcribe the type Ia 6-kb normal abl mRNA from the Philadelphia chromosome. However, we have found that the 6-kb transcript is present only in CML cell lines containing a normal abl allele and that the apparent inactivation of the nested Pa promoter is associated with allele-specific methylation. Furthermore, we have noticed that the Pa promoter is contained within a CpG island and undergoes progressive de novo methylation in the course of the disease. This is attested to by the fact that DNA samples from CML patients that are methylation-free at the time of diagnosis invariably become methylated in advanced CML. Since tumor progression in CML cannot always be inferred from the clinical presentation, assessment of de novo CpG methylation may prove to be of critical value in management of the disease. It could herald blastic transformation at a stage when bone marrow transplantation, the only potentially curative therapeutic procedure in CML, is still effective.

Animals↗
PubMed1994

Transcription of the insulin gene: towards defining the glucose-sensitive cis-element and trans-acting factors.

Previous work has shown that the sequence -196 to -247 of the rat insulin I gene mediates the stimulatory effect of glucose in fetal islets. We have used adult rat and human islets to delineate the glucose-sensitive cis-element to the sequence -193 to -227. In electrophoretic mobility shift assays, a 22 bp nucleotide corresponding to the sequence -206 to -227 bound all the nuclear proteins that could be bound by the entire minienhancer sequence -196 to -247. The rat insulin I sequence -206 to -227 formed three major complexes; in contrast, the corresponding human insulin sequence formed one single band with human and rat islet nuclear extracts, corresponding to the complex C1 of the rat insulin gene. Incubation of islets with varying glucose levels resulted in a dose-dependent increase in the intensity of the C1 band, while the other nuclear complexes formed with the insulin sequence, or the AP1 and SP1 binding activities used as control, were glucose insensitive. This is thus the first demonstration of a physiologic glucose-sensitive trans-acting factor for the insulin gene, whose further study may markedly enhance our understanding of the regulation of insulin biosynthesis in normal and diabetic beta cells. Furthermore, once cloned, the introduction of this glucose sensitive factor may enable the construction of truly physiologic artificial beta cells.

Animals↗
PubMed1993

Glucose modulates the binding of an islet-specific factor to a conserved sequence within the rat I and the human insulin promoters.

In cultured rat and human pancreatic islets, glucose stimulated transcription of the rat insulin I gene through the mini-enhancer (FF) located between residues -196 and -247. The glucose-sensitive element was delineated to the region -193 to -227. The mini-enhancer 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 of the human insulin promoter interacted with rat islet nuclear extracts to form a single complex, corresponding to the C1 complex of the rat insulin I sequence. C1 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 C1 complex corresponds to the glucose-sensitive factor and, consequently, may play a determining role in glucose-regulated expression of the insulin gene.

Animals↗