Molecular mechanisms underlying the glucose-dependent transcription of the insulin and glucokinase genes in the pancreatic beta-cell

Sammanfattning: Background: Insulin is of vital importance in the maintenance of the glucose homeostasis in mammals. This necessitates a tight regulation of both insulin release and biosynthesis. Although pancreatic beta-cells secrete only a fraction of the stored insulin upon glucose stimulation, insulin biosynthesis starts immediately in order to replenish the insulin store. It is well documented that glucose exerts its immediate effects at the posttranscriptional and translational levels by enhancing the stability of insulin-mRNA and up-regulating translation initiation and translation elongation. The glucose effect on transcription, however, is believed to occur only after several hours of exposure of the P-cell to high glucose. Because blood-glucose levels are only elevated for at most 2 hours after food uptake, we hypothesized the existence of a short-term control of gene transcription by glucose. Since the beta-cell transcription unit of the glucokinase gene has been shown to be regulated by the same stimuli as the insulin gene and has been discussed to utilize similar/same transcription regulatory elements, we postulated that also here glucose may affect transcription immediately. Aim: The aims of this study were 1.) to evaluate the existence of a glucose-dependent shortterm regulation of the transcription of the insulin and glucokinase genes and, if so, 2.) to identify the molecular mechanisms involved. Findings: By studying mRNA steady-state levels, transcription initiation and by establishing a technique that allowed us to monitor gene transcription online, we demonstrated, that insulin gene transcription is indeed regulated by short-term exposure of pancreatic beta-Cells to glucose. Investigation of the mechanisms underlying the short-term control revealed a link between insulin exocytosis and insulin gene transcription: insulin, secreted in response to glucose stimulation, up-regulates insulin gene transcription through a pathway that involves the insulin receptor/PI3 kinase/p70 s6 kinase and CaM kinases. Analysis of insulin promoter cis-elements showed the involvement of A- and E-boxes in glucose-dependent up-regulation of insulin gene transcription. Because the P-cell specific transcription factor PDX-1, which binds to A-boxes in the insulin promoter, has been discussed to be involved in glucose-dependent up-regulation of insulin gene transcription by a mechanism involving cytoplasmic-nuclear translocation, we aimed to identify the amino acid sequences responsible for the nuclear import of PDX-1. Sitedirected mutagenesis of putative phosphorylation sites and of positively charged amino acids in putative nuclear localization signal (NLS) motifs revealed that not a specific phosphorylation site, but the presence of the NLS motif RRMKWKK is necessary and, in conjunction with the integrity of the 'helix 3' domain of the PDX-1 homeodomain, sufficient for nuclear translocation of PDX-1. Analysis of the glucose-dependent regulation of glucokinase gene transcription showed, as is the case for the insulin gene, secreted insulin being a key factor. Surprisingly, the molecular mechanisms involved in insulin-stimulated glucokinase gene transcription are different from those of insulin-stimulated insulin gene transcription To study these differences in more detail we established a system that allowed us to monitor the activity of both the insulin and glucokinase promoters simultaneously within the same cell. By using this technique, we were able to demonstrate that insulin-stimulated insulin gene transcription is regulated through insulin receptor A/P13 kinase class la/p70 s6 kinase, whereas glucokinase transcription is activated by a mechanism involving insulin receptor B/P13 kinase class II like activity/PKB(c-Akt). Conclusion: We were able to demonstrate that both the insulin and the glucokinase genes are regulated by short-term exposure of pancreatic beta-cells to elevated glucose. The mechanism involves an insulin feedback loop, which utilizes different regulatory pathways for the activation of the two genes. Insulin gene transcription is activated by a mechanism involving insulin receptor A, P13 kinase class Ia and p70 s6 kinase. Glucokinase gene expression, however, is regulated through insulin receptor B, P13 kinase class II-like activity and PKB(c-Akt). These data demonstrate that selective insulin signaling can be gained through two different isoforms of the insulin receptor and report the first insulin receptor isoform-specific read-out. More importantly, the data demonstrate that the beta-cell is a target for insulin action. This implicates that beta-cell insulin resistance might contribute to the development of a betacell dysfunction in type 2 diabetes