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GAD65 and GAD67 are two isoforms of the enzyme glutamic acid decarboxylase which catalyze the production of GABA from glutamate, primarily in the brain. However, GAD and GABA also prevail in the retina, testes and islets of Langerhans. The main function of GABA is in neurotransmission, and it is involved in paracrine signalling in islets, but has also been suggested to play a role as a trophic factor in synaptogenesis and to be an important metabolite feeding into the tricarboxylic acid cycle via the GABA-shunt. Both GAD isoforms are subject to regulation, e.g. by synaptic activity. GAD65 is regulated at the level of enzyme activity by association and dissociation from its cofactor, PLP, whereas GAD67 is controlled at the level of its mRNA. To study this process in further detail, we have isolated and characterized the 5'-flanking region of the rat GAD67 gene. We report the transcriptional initiation sites and promoter sequences important for expression in islet beta-cells and C6 glioma cells, and demonstrate that the GAD67 promoter harbors elements that are responsive to glucose in primary islet cells.

The paired box and homeodomain containing transcription factors Pax4 and Pax6 are known to be essential for development of the pancreatic endocrine cells. In this report we demonstrate that stable expression of Pax4 in a rat glucagon-producing cell line inhibits the endogenously expressed glucagon gene completely. Furthermore, Pax4 represses Pax6 independent transcription of the insulin promoter, suggesting that Pax4 can actively repress transcription in addition to acting by competition with the transcriptional activator Pax6.

Glucose-stimulated expression of the insulin gene in β cells is mediated by the PDX-1 transcription factor. In this report, we show that stimulation results from effects on activation and DNA-binding potential. Thus, glucose specifically stimulated expression in MIN6 β cells from chimeras of PDX-1 and the GAL4 DNA-binding domain which spanned the N-terminal PDX-1 activation domain located between amino acids 1 to 79. GAL4:PDX activity was induced over physiological glucose concentrations and was also regulated by effectors of this response. The level of endogenous PDX-1 binding and phosphorylation were also induced under these conditions. We discuss how changes in PDX-1 phosphorylation may influence activity in glucose-treated β cells.

Insulin gene transcription is a unique feature of the pancreatic beta cells and is increased in response to glucose. The recent cloning of insulin promoter factor 1 (IPF1) and somatostatin transcription factor 1 (STF1) unexpectedly revealed that these are mouse and rat homologues of the same protein mediating transactivation through binding of CT box-like elements in rat insulin 1 and somatostatin promoter/enhancer regions, respectively. By using oligonucleotides representing each of the three CT boxes of the human insulin (HI) gene enhancer and nuclear extracts from the mouse islet tumor cell lines beta TC3 and alpha TC1, we have identified a beta-cell-specific binding activity as reported for IPF1, which has maximal affinity toward the CT2 box. However, in pluripotent, HI-transfected rat islet tumor cells, NHI-6F, this binding activity is present prior to induction of (human) insulin gene transcription. Its migration is identical to that of in vitro translated STF1 in electrophoretic mobility-shift assays; it is specifically recognized by anti-STF1 antibodies and has an apparent molecular mass of 46 kDa. Mutation of the CT2 box decreases transcriptional activity of a HI reporter plasmid by approximately 65% in beta TC3 cells and blocks the glucose response in isolated newborn rat islet cells. Furthermore, cotransfection with STF1 cDNA into the glucagon-producing alpha TC1 cells increases the activity of the HI enhancer 4- to 5-fold, suggesting that STF1/IPF1 can confer on alpha TC1 cells the ability to transcribe the HI gene. We conclude that STF1/IPF1 is a necessary but not sufficient key regulator of insulin gene activity, possibly also involved in glucose-regulated transcription.

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PDX-1 is a homeodomain transcription factor whose targeted disruption results in a failure of the pancreas to develop. Mutations in the human pdx-1 gene are linked to an early onset form of non-insulin-dependent diabetes mellitus. PDX-1 binds to and transactivates the promoters of several physiologically relevant genes within the beta-cell, including insulin, glucose transporter 2, glucokinase, and islet amyloid polypeptide. This study focuses on the mechanisms by which PDX-1 activates insulin gene transcription. To evaluate the role of PDX-1 in transcription of the insulin gene, a chloramphenicol acetyltransferase reporter construct was designed with a single yeast GAL4-DNA binding site in place of the A3/PDX-1 binding element in the rat insulin II enhancer. In the presence of GAL4:PDX chimeras containing N-terminal transactivation domain sequences, this GAL4-substituted insulin construct was active in PDX-1-expressing beta-cells and not non-beta-cells. PDX-1 activation was mediated through three highly conserved segments of the transactivation domain. In addition, when cotransfected together with the GAL4-substituted insulin enhancer reporter gene in glucose-responsive MIN-6 beta-cells, glucose-induced activation is observed with GAL4:PDX-1 but not with fusions of the heterologous activation domains from herpes virus VP16 or adenovirus-5 E1A proteins. Using A3 element-substituted GAL4 insulin enhancer reporter constructs containing mutations in two additional key control elements, E1 and C1, we also show that full activation requires cooperative interactions between other enhancer-bound factors, particularly the E1 element activators. In contrast, the activity of the VP16 activation factor was not dependent on the activators of either the E1 or C1 sites. These results suggest that the PDX-1 transactivation domain is specifically required for appropriate regulation of insulin enhancer function in beta-cells.

α-cell specific transcription of the glucagon gene is mainly conferred by the glucagon promoter G1-element, while additional elements G2, G3, and G4 have broad islet cell specificity. Transcription of the glucagon gene has been shown to be stimulated by Pax6 through binding to the glucagon gene promoter G3-element. In this report, we show that Pax6 additionally binds the glucagon gene promoter G1-element and forms a transcriptionally active complex with another homeodomain protein, Cdx2/3. Two distinct mutations in the G1-element, that both reduce promoter activity by 85–90%, is shown to eliminate binding of either Pax6 or Cdx2/3. Additionally, Pax6 enhanced Cdx2/3 mediated activation of a glucagon reporter in heterologous cells. We discuss how Pax6 may contribute to cell-type specific transcription in the pancreatic islets by complex formation with different transcription factors.

Electrophoretic mobility shift assays were performed using oligonucleotides corresponding to known protein binding sites within the human insulin gene enhancer and nuclear extracts from mouse pancreatic alpha and beta cell lines. The results demonstrate that a previously described factor, IUF-1, binds to three sites at -82 (the CT1 box), -215 (the CT2 box), and -319 (the CT3 box) in the human insulin gene enhancer. IUF-1 was present only in beta but not in alpha cells, while all other DNA-binding proteins were present in both cell lines. IUF-1 may therefore be an important determinant of insulin gene beta cell-specific expression.

The somatostatin upstream enhancer (SMS-UE) is a highly complex enhancer element. The distal A-element contains overlapping Pdx1 and Pbx binding sites. However, a point mutation in the A-element that abolishes both Pdx1 and Pbx binding does not impair promoter activity. In contrast, a point mutation that selectively eliminates Pdx1 binding to a proximal B-element reduces the promoter activity. The B-element completely overlaps with a Pax6 binding site, the C-element. A point mutation in the C-element demonstrates that Pax6 binding is essential for promoter activity. Interestingly, a block mutation in the A-element reduces both Pax6 binding and promoter activity. In heterologous cells, Pdx1 potentiated Pax6 mediated activation of a somatostatin reporter. We conclude that the β/δ-cell-specific activity of the SMS-UE is achieved through simultaneous binding of Pdx1 and Pax6 to the B- and C-elements, respectively. Furthermore, the A-element appears to stabilise Pax6 binding.

The homeodomain (HD) protein Nkx6.1 is the most beta-cell-specific transcription factor known in the pancreas and its function is critical for the formation of the insulin-producing beta-cells. However, the target genes, DNA-binding site, and transcriptional properties of Nkx6.1 are unknown. Using in vitro binding site selection we have identified the DNA sequence of the Nkx6.1 binding site to be TTAATTG/A. A reporter plasmid containing four copies of this sequence is activated by an Nkx6.1HD/VP16 fusion construct. Full-length Nkx6.1 fails to activate this reporter plasmid in spite of robust interaction with the binding site in vitro. Stable expression of Nkx6.1 in the glucagon-producing alpha-cell-like MSL-G-AN cells induces expression of the endogenous insulin gene in a subset of the cell population. The expression of other known beta-cell-specific factors such as Pax4, Pax6, Pdx1, GLUT2 and GLP1-R is unchanged by the introduction of Nkx6.1.

The helix–loop–helix transcription factor NeuroD1 (also known as Beta2) is involved in β-cell survival during development and insulin gene transcription in adults. Here we show NeuroD1 is primarily cytoplasmic at non-stimulating glucose concentrations (i.e. 3 mM) in MIN6 β-cells and nuclear under stimulating conditions (i.e. 20 mM). Quantification revealed that NeuroD1 was in 40–45% of the nuclei at 3 mM and 80–90% at 20 mM. Treatment with the MEK inhibitor PD98059 or substitution of a serine for an alanine at a potential mitogen-activated protein kinase phosphorylation site (S274) in NeuroD1 significantly increased the cytoplasmic level at 20 mM glucose. The rise in NeuroD1-mediated transcription in response to glucose also correlated with the change in sub-cellular localization, a response attenuated by PD98059. The data strongly suggest that glucose-stimulation of the MEK–ERK signalling pathway influences NeuroD1 activity at least partially through effects on sub-cellular localization.