Danielle Melloul

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

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

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.

Autoregulation of glucose transport: effects of glucose on glucose transporter expression and cellular location in muscle.

Decreased peripheral utilization of glucose is an important pathogenic mechanism in diabetes. Although it is universally acknowledged that insulin resistance plays a major role in the reduction of glucose consumption by peripheral tissues, and many defects have been described in the function of insulin receptors (see chapter by Olefsky), indirect clinical and in vivo experimental observations suggest that hyperglycemia per se may participate in inducing and/or maintaining a reduced glucose uptake (summarized in chapter by De Fronzo). The idea occurred to us some years ago that a certain analogy may exist between the downregulation of hormone receptors by augmented hormone concentrations, and the reduction of glucose uptake by hyperglycemia. The extraordinary redundancy of compensatory events that operate in vivo make the testing of such a hypothesis near-impossible. We therefore chose to work in vitro, and because muscle is the main glucose consumer of the periphery, we focused on in vitro muscle preparations and myocyte lines.

Chronic exposure to high glucose impairs multiple β-cell functions in cultured human pancreatic islets

Deficient insulin secretion and relative hyperproinsulinemia are characteristic of non–insulin-dependent diabetes. Culture of human islets at high glucose concentrations generates β-cell dysfunctions that mimic those observed in diabetic patients, with depleted islet insulin stores. The increased proinsulin ratio probably results from accelerated discharge of insulin granules, rather than diminished expression of convertase. Insulin mRNA levels are decreased, and the rate of transcription of the human insulin gene is suppressed. In human islets exposed to high glucose for 4 to 9 days, the DNA binding of PDX-1, the key transcriptional factor of the insulin gene, is reduced, as is its mRNA level. These effects are reversed by normalizing the culture glucose concentration. It is proposed that β-cell “glucotoxicity” results from the inability of proinsulin biosynthesis to keep pace with chronic insulin hypersecretion. At least in human β-cells, downregulation of PDX-1 expression by hyperglycemia is the main cause for depletion of the insulin stores.