Rat Insulin Gene Research Articles

Thymic epithelial cell–specific deletion of Jmjd6 reduces Aire protein expression and exacerbates disease development in a mouse model of autoimmune diabetes

Thymic epithelial cells (TECs) establish spatially distinct microenvironments in which developing T cells are selected to mature or die. A unique property of medullary TECs is their expression of thousands of tissue-restricted self-antigens that is largely under the control of the transcriptional regulator Aire. We previously showed that Jmjd6, a lysyl hydroxylase for splicing regulatory proteins, is important for Aire protein expression and that transplantation of Jmjd6-deficient thymic stroma into athymic nude mice resulted in multiorgan autoimmunity. Here we report that TEC-specific deletion of Jmjd6 exacerbates development of autoimmune diabetes in a mouse model, which express both ovalbumin (OVA) under the control of the rat insulin gene promoter and OT-I T cell receptor specific for OVA peptide bound to major histocompatibility complex class I Kb molecules. We found that Aire protein expression in mTECs was reduced in the absence of Jmjd6, with retention of intron 2 in Aire transcripts. Our results thus demonstrate the importance of Jmjd6 in establishment of immunological tolerance in a more physiological setting.

Retrovirus vector transfection of rat insulin gene into pancreas decrease blood glucose of diabetic rat

Human and animal diabetes mellitus were controlled by a dietary treatment supplemented with either a sulfonylurea drug or insulin injection. Insulin injections were inconvenient and the hypoglycemia induced by insulin-overdose could be fatal. Sulfonylurea drugs were administered orally, however, do not typically provide satisfactory control of blood glucose as a starting treatment in 25% - 30% patients. Therefore, it was imperative to develop a method for the control of human and animal diabetes mellitus. Recently, insulin gene transferred and expressed in non-pancreatic cells as a means for the treatment of diabetes was developed rapidly in the expanding gene therapy. Retrovirus, lentivirus, adenovirus, adenoassociated virus and herpes simplex had been used as viral vectors, and the constructed viral-insulin gene was successfully transferred into diabetic rat cells. A gene, containing promoter, enhancer and rat type I insulin gene (a-chain, b-chain and signal peptide), was constructed into a retrovirus vector in the study. The constructed viral-insulin gene was transferred into mouse fibroblast cell. The insulin concentration in 3-day cultured mouse fibroblast cells was 4806.35 ± 53.72 pg/ml. The insulin concentration for the viral vector containing enhancer and promoter of rat insulin gene was higher than the vector containing only insulin gene by a 61% increase in the cultured mouse fibroblast cells. The enhancer and promoter activity of rat insulin gene would be an important determinant for the expression of insulin gene. The secreted amount of insulin by retrovirus vector contained enhancer/promoter gene in this study could achieve as high concentrations (4806.35 ± 53.72 pg/ml) as the insulin injection therapy. Blood glouse decreased sig- nificantly for at last 10 days demonstrated that transfection, direction injection of viral-insulin gene into pancreas of diabetic rat, was successful. These studies suggest that the retrovirus vector might be used to transfer the insulin gene in vitro and in vivo.

The Activation of the Rat Insulin Gene II by BETA2 and PDX-1 in Rat Insulinoma Cells Is Repressed by Pax6

The transcriptional transactivator Pax6 binds the pancreatic islet cell-specific enhancer sequence (PISCES) of the rat insulin I gene. However the human, mouse, and rat insulin gene II promoters do not contain a PISCES element. To analyze the role of Pax6 in those PISCES-less promoters, we investigated its influence on rat insulin gene II expression and included in our studies the main activators: pancreatic and duodenal homeobox protein-1 (PDX-1) and BETA2/E47. Luciferase assays, Northern blots, and RIA were used to study effects of Pax6 overexpression, gel shift and chromatin precipitation assays to study its binding to the DNA, and yeast two-hybrid assays and glutathione S transferase capture assays to investigate its interactions with PDX-1 and BETA2. Finally, glucose-dependent intracellular transport of Pax6 was demonstrated by fluorescence microscopy. Overexpression of Pax6 prevents activation of the rat insulin II gene by BETA2 and PDX-1 and hence suppresses insulin synthesis and secretion. In vitro, Pax6 binds to the A-boxes, thereby blocking binding of PDX-1, and at the same time, its paired domain interacts with BETA2. Fluorescence microscopy demonstrated that the nuclear-cytoplasmic localization of Pax6 and PDX-1 are oppositely regulated by glucose. From the results, it is suggested that at low concentrations of glucose, Pax6 is localized in the nucleus and prevents the activation of the insulin gene by occupying the PDX-1 binding site and by interacting with BETA2.

In Vivo Detection of Extrapancreatic Insulin Gene Expression in Diabetic Mice by Bioluminescence Imaging

BackgroundExtrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP).MethodsThe Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis.ResultsStreptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia.ConclusionsBLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.

The LIM-Homeodomain Protein ISL1 Activates Insulin Gene Promoter Directly through Synergy with BETA2

The LIM-homeodomain transcription factor ISL1 (islet factor 1) is essential for pancreatic islet cell and dorsal mesenchyme development. Mutations in ISL1 are associated with maturity-onset diabetes of the young and type 2 diabetes. Whether ISL1 plays a role in the insulin gene expression has not been fully elucidated. In the present study, we show that ISL1 can synergistically activate insulin gene transcription with BETA2 in pancreatic β cells. The protein–protein interactions of ISL1 and BETA2 are directly mediated by the LIM domains of ISL1 and the basic helix–loop–helix domain of BETA2. Deletion of the two LIM domains of ISL1 enhances the transcriptional activation of the insulin gene, indicating a key role for the homeodomain in activating the insulin promoter. Furthermore, ISL1 can bind with the A3/4 box in the rat insulin gene І promoter through its homeodomain. ISL1 expression is up-regulated at the mRNA level in type 2 diabetes (db/db mouse model) but down-regulated by dexamethasone in rat insulinoma cells. These results suggest that ISL1 is a transcriptional activator for insulin gene expression, and the interactions of ISL1 with BETA2 are required for the transcriptional activity of the insulin gene. Reduction in Isl1 gene expression appears to be involved in the impairment of insulin expression mediated by dexamethasone.

Tacrolimus‐Induced Diabetes in Rats Courses with Suppressed Insulin Gene Expression in Pancreatic Islets

An animal model of post-transplant diabetes was induced in rats by treating them daily with 0.1 mg/kg body weight of tacrolimus (FK506) in two i.p. injections. Rats developed hyperglycaemia and glucose intolerance after 9 days of treatment. Pancreatic islets, isolated from treated rats on different days, showed a decreased capacity to secrete insulin in response to 20 mM glucose at days 7 and 14. This suppression of insulin secretion was preceded by a reduction of the islet insulin content on day 5 that was progressively decreasing until the end of the treatment (day 14). Islet content of insulin mRNAs, transcribed from rat insulin genes 1 and 2, was strongly suppressed, similar to the insulin content, at days 7 and 14. Islet mass was not strikingly modified by tacrolimus treatment: the DNA content was slightly decreased at the end (day 14) and the rate of islet cell apoptosis slightly increased. Tacrolimus-induced diabetes in the rat seems to be mainly provoked by a decreased insulin gene transcription with little or no alteration of islet mass. This explains that the observed suppression of all the islet and animal parameters studied was completely reversed 2 weeks after interrupting tacrolimus treatment.

Engineered Enteroendocrine Cells Secrete Insulin in Response to Glucose and Reverse Hyperglycemia in Diabetic Mice

Type 1 diabetes is a metabolic disorder caused by loss of insulin-producing pancreatic beta-cells. Expression of insulin in non-beta-cells to create beta-cell surrogates has been tried to treat type 1 diabetes. Enteroendocrine K cells have characteristics similar to pancreatic beta-cells, such as a glucose-sensing system and insulin-processing proteases. In this study, we genetically engineered an enteroendocrine cell line (STC-1) to express insulin under the control of the glucose-dependent insulinotropic polypeptide promoter. We screened clones and chose one, Gi-INS-7, based on its high production of insulin. Gi-INS-7 cells expressed glucose transporter 2 (GLUT2) and glucokinase (GK) and secreted insulin in response to elevated glucose levels in vitro. To determine whether Gi-INS-7 cells can control blood glucose levels in diabetic mice, we transplanted these cells under the kidney capsule of streptozotocin (STZ)-induced diabetic mice and found that blood glucose levels became normal within 2 weeks of transplantation. In addition, glucose tolerance tests in mice that became normoglycemic after transplantation with Gi-INS-7 cells showed that exogenous glucose was cleared appropriately. These results suggest that engineered K cells may be promising surrogate beta-cells for possible therapeutic use for the treatment of type 1 diabetes.

997. Specific Expression of Transgenes in Insulin-Secreting Islet Cells by Lentiviral Vectors

Gene transfer into pancreatic islet cells is a basis requirement for gene therapy approaches to insulin-dependent diabetes (IDDM). Lentiviral vectors carrying the GFP gene under the control of ubiquitously expressing promoters like the cytomegalovirus (LV-CMV-GFP) or the phosphoglycerate kinase (LV-PGK-GFP) promoter efficiently transduce porcine Langerhans islets and express the transgene. However, apart from insulin-secreting B cells, three other cell types exist in pancreatic islets: A (glucagon-producing) cells, D (somatostatin-secreting) and F (pancreatic polypeptide-producing) cells. To achieve specific transgene expression in insulin-secreting cells, we tested different promoters. Two nonallelic genes encoding preproinsulin have been described for mouse and rat. We cloned promoter sequences of the rat insulin I (RIPI, 420bp) and insulin II (RIPII, 600bp) gene into an HIV-1 derived lentiviral vector 5' of the GFP gene, resulting in LV-RIPI-GFP and LV-RIPII-GFP, respectively. Rat islet cell line (RINm5f) cells were infected with an identical amount of p24 of LV-RIPI-GFP, LV-RIPII-GFP, and LV-CMV-GFP. Fluorescence-activated cell sorting (FACscan) revealed GFP expression in 69,4% of the LV-RIPI-GFP infected cells, whereas only 42,1% of the LV-RIPII-GFP infected RINm5f cells expressed GFP. Comparison of the mean fluorescence revealed a 2.5 higher expression level (52,7 versus 19,3) in LV-RIPI-GFP-infected cells, as compared to the LV-RIPII-GFP infected cells. 98,4% of the LV-CMV-GFP infected RINm5f cells were GFP-positive with a mean fluorescence of 4267. As controls we infected porcine fetal fibroblasts with the lentiviral vectors, and observed a ~ 4-fold reduction in the number of GFP-positive cells in both LV-RIPI-GFP and LV-RIPII-GFP. In contrast, the number of GFP-positive fibroblasts after infection with LV-CMV-GFP was not significantly reduced as compared to the RINm5f cells. Given the high similarities of human and porcine anatomy and physiology, diabetes models in the pig would be much more clinically relevant than rodent models. In addition, the porcine pancreas could be a source of islet xenografts for IDDM patients. Therefore, we are currently investigating the use of lentiviral vectors to achieve B-cell specific transgene expression in vivo in the porcine pancreas. Lentiviral vectors efficiently transduce islet cells, and insulin-specific expression can be achieved using rat insulin gene promoters, with RIPI resulting in a higher expression levels in insulin-producing cells than RIPII.

Cloning and analysis of axolotl ISL2 and LHX2 LIM-homeodomain transcription factors.

We cloned and characterized the ISL2 and LHX2 LIM-homeodomain transcription factors of the Mexican salamander, or axolotl, Ambystoma mexicanum. Using a degenerate PCR approach, partial cDNAs representing five LIM-homeodomain genes were cloned, indicating conservation of this class of transcription factors in urodeles. Full-length cDNAs for Isl2 and Lhx2 were identified and sequenced. The predicted ISL2 and LHX2 proteins are well conserved, especially in the LIM and DNA-binding domains. The Isl2 and Lhx2 genes are expressed at all examined stages of embryogenesis and display tissue-restricted expression patterns in adults. In functional tests, axolotl LHX2 was inactive compared to homologous mammalian factors and adopted unusual DNA/protein complexes. However, axolotl ISL2 bound and induced transcription from the rat insulin gene. These experiments demonstrate conservation of key developmental regulatory proteins in salamanders and will allow future studies of their potential roles in the molecular regulation of tissue regeneration in such species.

Daily nasal inoculation with the insulin gene ameliorates diabetes in mice

The present study examined the feasibility of liposome-mediated gene transfer via nasal administration, for treating insulin-dependent diabetes mellitus. The rat insulin gene was packed under control of the CMV promoter, complexed with DC-chol/DOPE-based liposomes and administered daily via the nasal route in mice made severely diabetic by streptozocin. Sustained expression of the insulin gene was achieved and insulinopenia, ketonuria and death were prevented. Hyperglycemia and body weight reduction were significantly suppressed without evidence of hypoglycemia throughout the experimental period. RT-PCR and FISH analysis indicated that insulin was produced in the alveolar epithelial cells of the lung. Liposome-mediated in vivo gene transfer via nasal administration may provide an efficacious route for delivery of hormonal and other gene products into the blood stream.

The multiple endocrine neoplasia type 1 gene product, menin, inhibits insulin production in rat insulinoma cells.

A mutant gene isolated from a patient with multiple endocrine neoplasia type 1, MEN1 encodes a protein, menin. Features of MEN1 include multiple endocrine tumors of the parathyroid glands, anterior pituitary and pancreatic islets. Insulinoma, arising from the pancreas is the most common MEN1-related tumor. Menin is a nuclear protein and interacts with the transcription factor, Jun D, but whether menin has a role in the pathogenesis of MEN1-related endocrine tumors including insulinoma remains unknown. In this study, we examined the effects of menin on production of human insulin. Insulinoma cells, INS-1 were co-transfected with a vector that expressed menin and a reporter template containing 235 bp of the rat insulin gene 5'-flanking region fused to the luciferase gene to yield pINS-LUC. Promoter activity of the insulin gene was significantly decreased in co-transfected cells as compared to mock-transfected controls. INS-1 cells stably transfected with a vector that expressed menin were used to examine the insulin secretion. In cells with a high level of menin expression, both insulin secretion and thymidine incorporation into DNA were inhibited when compared to mock-transfected cells. Additionally, the rate of apoptosis of menin-transfected cells was increased compared to mock-transfected cells. These observations suggest that menin inhibits insulin promoter activity and secretion, and also cell proliferation, raising the possibility that menin may play an important role in the pathogenesis of insulinoma.

Over-expression of the glucagon-like peptide-1 receptor on INS-1 cells confers autocrine stimulation of insulin gene promoter activity: a strategy for production of pancreatic beta-cell lines for use in transplantation.

To develop transplantable beta-cell lines for the treatment of diabetes mellitus, we have taken advantage of the property of INS-1 cells to synthesize and secrete not only insulin, but also small quantities of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). In INS-1 cells over-expressing the beta-cell GLP-1 receptor (GLP-1-R), we have shown, by radioimmune assay and bioassay of conditioned medium, that an autocrine signaling mechanism of hormone action exists whereby self-secreted GLP-1 acts as a competence factor in support of insulin gene transcription. INS-1 cells also exhibit insulin gene promoter activity, as assayed in cells transfected with a rat insulin gene I promoter-luciferase construct (RIP1-Luc). The GLP-1-R agonist exendin-4 stimulates RIP1-Luc activity in a glucose-dependent manner, an effect mediated by endogenous GLP-1-Rs, and is blocked by the serine/threonine protein kinase inhibitor Ro 31-8220. Over-expression of GLP-1-R in transfected INS-1 cells reduces the threshold for exendin-4 agonist action, whereas basal RIP1-Luc activity increases 2.5-fold in the absence of added agonist. The increase of basal RIP1-Luc activity is a consequence of autocrine stimulation by self-secreted GLP-1 and is blocked by introduction of (1) an inactivating W39A mutation in the N-terminus ligand-binding domain of GLP-1-R or (2) mutations in the third cytoplasmic loop that prevent G protein coupling. No evidence for constitutive ligand-independent signaling properties of the GLP-1-R has been obtained. Over-expression of GLP-1-R increases the potency and efficacy of D-glucose as a stimulator of RIP1-Luc. Thus, INS-1 cells over-expressing the GLP-1-R recapitulate the incretin hormone effect of circulating GLP-1, thereby providing a possible strategy by which beta-cell lines may be engineered for efficient glucose-dependent insulin biosynthesis and secretion.

Defective adenoassociated viral-mediated transfection of insulin gene by direct injection into liver parenchyma decreases blood glucose of diabetic mice.

The present study assessed the feasibility of transferring the insulin gene into liver cells of diabetic individuals using a defective adenoassociated viral (AAV) vehicle. AAV offers several advantages over other viral vectors, since this vehicle can facilitate transfection in vivo without cell division and without any viral coding sequences (thus minimizing inflammation). The rat insulin gene and lacZ were each packed into a defective AAV vehicle (AAV-INS and AAV-lacZ, respectively). Successful AAV-mediated transfection and expression of lacZ into hepatocytes in primary cell culture were demonstrated by chemiluminescent assay of beta-galactosidase. Similarly, AAV-mediated transfection and expression of the insulin gene into hepatocytes was demonstrated by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). After AAV-mediated transfection of the insulin gene into hepatocytes, glucose in the medium was significantly reduced for up to 5 days. After direct injection of AAV-INS into liver parenchyma of diabetic mice, successful transfection was demonstrated by RT-PCR, and blood glucose was significantly decreased for at least 6 days. These studies suggest that the AAV vector may be used to transfer the insulin gene into liver cells in vitro and in vivo.

Courts Take a Narrow View of UC's Claims

SCIENTIFIC COMMUNITYA 7-year patent dispute between the University of California and Eli Lilly and Company over rights to the insulin gene could prove troublesome for companies and researchers making broad claims on genes. The courts have ruled that a patent based on a landmark discovery of the rat insulin gene in 1977 does not cover the production of human insulin.

The Pan basic helix-loop-helix proteins are required for insulin gene expression.

The rat I insulin enhancer contains two principal regulatory elements, the Nir and Far motifs of an identical 9-base pair sequence, which function both as positive and negative cis-acting elements. The Nir and Far elements are targets for DNA-binding proteins, which play a predominant role in the selective transcription of the insulin gene in endocrine beta-cells. In vitro DNA-binding studies have demonstrated the ability of several helix-loop-helix (HLH) proteins, including the Pan/E2A proteins, upstream stimulating factor, human beta-HLH factor (rat beta-HLH factor), and E2-2/ITF-2, to bind the Nir and Far enhancer motifs. The presence of the aforementioned different HLH proteins in endocrine beta-cells, all of which display similar binding affinities for the Nir and Far elements in vitro, raises the question of which HLH proteins actively participate in the transcriptional regulation of the rat insulin I gene in pancreatic endocrine beta-cells. To investigate the specific role that Pan proteins play in regulating insulin gene expression, we have created endocrine beta-cell stable integrants that constitutively express Pan antisense transcripts that selectively inhibit endogenous Pan protein synthesis in differentiated beta-cells. We demonstrate that diminished Pan protein levels in beta-cells, caused by Pan antisense transcripts, accompanies a dramatic attenuation of rat insulin gene transcription. We also show that the decrease in Pan protein expression correlates with a specific reduction of the beta-endocrine-specific Nir and Far element-binding activity, insulin enhancer factor 1.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The LIM domain homeobox gene isl-1: conservation of human, hamster, and rat complementary deoxyribonucleic acid sequences and expression in cell types of nonneuroendocrine lineage.

The LIM domain homeobox gene isl-1 was originally isolated by virtue of its ability to bind DNA sequences from the 5'-flanking region of the rat insulin gene. Initial experiments localized isl-1 to the islet cells of the pancreas, suggesting a possible role for this protein in the regulation of insulin gene expression and/or islet cell development. More recent studies have determined that isl-1 is also expressed in the central and peripheral nervous system. We now report that isl-1 gene expression is not restricted to cells of neuroendocrine lineage. Northern blot analysis of rat and hamster fibroblasts, rat keratinocytes, and human epidermoid carcinoma cells detected messenger RNA transcripts that hybridized, at high stringency, to different isl-1 complementary DNA probes. DNA sequence analysis of human and hamster isl-1 complementary DNAs generated by reverse transcription-polymerase chain reaction from nonneuroendocrine cell lines demonstrated 100% conservation of isl-1 amino acid sequences from rat hamster and human species. Western blot analysis with isl-1 antiserum demonstrated that immunoreactive isl-1 was detectable in nuclear extracts from islet cell lines. In contrast, immunoreactive isl-1 was only detected in cytoplasmic extracts from nonneuroendocrine cell lines. The results of these studies demonstrate striking conservation of mammalian isl-1 coding sequences and widespread expression of the isl-1 gene in heterologous cells. These experiments suggest that the biological importance of isl-1 may not be restricted to neuroendocrine cell lineages and raise the possibility that isl-1 function may be regulated by sequestration in distinct cellular compartments.

The role of the proximal CTAAT-box of the rat glucokinase upstream promoter in transcriptional control in insulin-producing cells.

Sequence analysis of the 5'flanking region of the beta-cell specific transcription unit of the rat glucokinase gene (r beta GK) revealed the presence of sequence motifs very similar to the IEB-(Far)-box and a CT-motif which play a crucial role in transcriptional control of insulin genes. 5'deletional analysis of the r beta GK proximal promoter element (localized between nucleotides -278 and -49) as well as site directed mutagenesis showed that both motifs are mutationally sensitive and contribute to transcriptional control in HIT M2.2.2 cells. The combination of the IEB-(Far)-like motif with the CT-box was unable to form a "mini-enhancer" similar to the Far-FLAT-element of the rat insulin I gene promoter but rather functions as a beta-cell specific control element in r beta GK expression. Electrophoretic mobility shift assays (EMSAs) and competition studies using oligonucleotides containing CT-motifs of rat insulin genes promoters, human insulin gene promoter, and rat amylin gene promoter showed similar binding patterns with nuclear extracts isolated from insulin-producing cell lines. These studies indicate that CT-motifs of rat glucokinase, insulin, and amylin gene promoters may bind similar--probably identical--nuclear factor(s) and may play a central role in the coordinated expression of these genes in insulin-producing cells.

An insulinoma nuclear factor binding to GGGCCC motifs in human insulin gene.

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on 5' flanking DNA elements. In the enhancer of rat I insulin gene, two elements, the Nir and Far boxes, located at positions -104 and -233 respectively and containing the same octameric motif are essential for B cell specific transcription activity. Homologous sequences are present in the human insulin gene. While studying the binding of nuclear proteins from insulinoma cells to the -258/+241 region of the human insulin gene, we observed a previously undetected protein binding site in the intron I region between nucleotides +160 and +175. The binding activity was present in insulin producing cells such as RIN and HIT insulinoma cells but not in fibroblasts or insulin negative fibroblast x RIN hybrid cells. DNAse I footprinting and gel retardation/methylation interference experiments allowed us to define the core binding site of the intron binding factor as a GGGCCC hexamer. This factor is also capable to bind to a related sequence, contiguous to the Far-like element in rat and human insulin genes. The binding of the GGGCCC binding factor in this critical region of the insulin gene enhancer may participate in the regulation of insulin gene expression.

Human glucokinase gene: isolation, structural characterization, and identification of a microsatellite repeat polymorphism.

The gene encoding human glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a major component of glucose sensing in pancreatic islet beta-cells, was isolated and characterized. The gene was shown by Southern blotting to exist as a single copy in the genome which mapped to chromosome 7p. It contained 12 exons including two tissue-specific first exons, one active in islet beta-cells (1B), and the other active in liver (1H), and one optional cassette exon which was expressed as a minor form in the liver. Thus the three previously reported isoforms of glucokinase mRNA were the result of tissue-specific activation of separate liver and islet promoters and subsequent alternative splicing events. Eleven exons, including 1H and the optional cassette exon 2A, were scattered over 16 kilobase (kb) in the genome, while exon 1B was separated from the rest by at least 20 kb. Although the islet promoter was found to lack a TATA box, a major transcript from the islet promoter was mapped 486 nucleotides upstream of the translation initiation site. The presence in the islet glucokinase promoter of the potential control element GCCACCAG, a homology of the regulatory element present in both human insulin (GCCACCGG) and rat insulin (GCCATCTG) genes, implied a possible tissue-specific regulatory role of this element. The liver promoter was found to contain a TATA box-like sequence, and transcription was initiated predominantly at 168 nucleotides upstream of the translation initiation site of the major isoform. A new highly polymorphic microsatellite, composed of a compound imperfect dinucleotide repeat [GT]15[GA]8CA[GA]7CA[GA]3AA[GA]2, was mapped 6 kb upstream of islet exon 1. A polymerase chain reaction-based assay was developed, and seven different sized alleles were identified in American Blacks. The sequence information reported here, along with the new polymorphic marker, will make it possible to clarify the molecular basis of potential glucokinase defects in noninsulin-dependent diabetes mellitus patients and may further elucidate the nature of genetic susceptibility to the development of this common metabolic disease.