Regulation Of Insulin Gene Expression Research Articles

Identification of a single nucleotide insertion polymorphism in the upstream region of the insulin promoter factor-1 gene: an association study with diabetes mellitus.

Insulin promoter factor 1 (IPF1) is a key factor both for the regulation of insulin gene expression and for the development of the pancreas. In this study 88 patients with non-insulin-dependent diabetes mellitus (NIDDM) who were diagnosed as diabetic at less than 40 years of age, 55 patients with insulin-dependent-diabetes (IDDM), and 67 normal control subjects were analysed for variants in the upstream region of the IPF1 gene by direct sequencing. A novel single nucleotide insertion polymorphism was found in a guanine triplet at 108 bp upstream of the translation start site. The G insertion allele (G4 allele) was found to be common in the Japanese population, at a frequency of 0.50. The prevalence of G3 homozygotes was higher in IDDM patients (35%) and lower in NIDDM patients (17%) than in normal control subjects (28%, p=0.049). In the NIDDM group, the ratio of insulin treatment tended to be higher in subjects homozygous for the G3 allele, although the genotype was not significantly associated with basal C-peptide levels. The polymorphism is unlikely to be a major contributor to the insulin deficiency of diabetes. However, the polymorphic locus, or an unknown mutation which is in linkage disequilibrium with the polymorphism, could be involved in the pathophysiology of diabetes. The high heterozygosity may be useful for genetic linkage studies of other mutations within and near the IPF1 gene.

Effects of tacrolimus (FK506) on human insulin gene expression, insulin mRNA levels, and insulin secretion in HIT-T15 cells.

FK506 (tacrolimus) is an immunosuppressive drug which interrupts Ca2+-calmodulin-calcineurin signaling pathways in T lymphocytes, thereby blocking antigen activation of T cell early activation genes. Regulation of insulin gene expression in the beta cell may also involve Ca2+-signaling pathways and FK506 has been associated with insulin-requiring diabetes mellitus during clinical use. The purpose of this study was to characterize the effects of FK506 on human insulin gene transcription, insulin mRNA levels, and insulin secretion using as a model the HIT-T15 beta cell line. FK506 had no acute effect on insulin secretion in the HIT cell, but caused a reversible time- and dose-dependent (10(-9)-10(-6) M) decrease in HIT cell insulin secretion. Decreased insulin secretion in the presence of FK506 was also accompanied by a dose-dependent decrease in HIT cell insulin content, insulin mRNA levels, and expression of a human insulin promoter-chloramphenicol acetyl transferase (CAT) reporter gene. FK506 decreased HIT cell expression of the human insulin promoter-CAT reporter gene by 40% in the presence of both low (0.4 mM) at high (20 mM) glucose concentrations. Western blot analysis of HIT cell proteins gave evidence for the presence of calcineurin in the HIT cell. These findings suggest that FK506 may have direct effects to reversibly inhibit insulin gene transcription, leading to a decline in insulin mRNA levels, insulin synthesis, and ultimately insulin secretion.

Insulin regulation of multiple ribonucleic acid species in human skeletal muscle in insulin-sensitive and insulin-resistant subjects.

In vivo short term (2 h) insulin-regulated gene expression was examined in skeletal muscle of persons with differing insulin sensitivities. Nine genes were analyzed by a S1 nuclease protection assay with multiple probes (multiple S1 nuclease protection assay) to allow the simultaneous examination of RNA abundances from the multiple genes. In insulin-sensitive individuals, 5 of these 9 genes were insulin responsive. RNA from the proto-oncogenes c-Ha-ras, c-myc, and c-src transiently increased 2- to 4-fold within 30 min of insulin infusion. In addition, the RNA abundance of myf-5, a muscle specific differentiation factor, increased 3-fold with a time course similar to that of c-Ha-ras, c-myc, and c-src. In contrast, type 1 protein phosphatase alpha (PPP1A) RNA levels decreased by 50% within 30 min. In insulin-resistant individuals, the RNA levels of c-Ha-ras and myf-5 did not increase, whereas c-src RNA did increase within 30 min of insulin infusion. RNA encoding c-myc transiently increased in both groups; however, this response was lower in insulin-resistant individuals than in insulin-sensitive individuals in a pattern similar to c-Ha-ras and myf-5. PPP1A RNA levels slightly increased in insulin-resistant individuals. In both insulin-sensitive and insulin-resistant persons, RNA quantities of GLUT4, c-jun, c-fos, and the insulin receptor did not change over the period of insulin infusion. However, overall RNA levels of the insulin receptor and c-jun were lower in insulin-resistant individuals.

The reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to high glucose concentration is associated with the loss of RIPE3b1 and STF-1 transcription factor expression.

The most important regulator of insulin gene expression in pancreatic beta- cells is glucose, which affects gene transcription, mRNA translation, and secretion. Insulin gene transcription is both positively and negatively regulated by glucose. Recently, we have shown that the inhibition of insulin gene transcription caused by passaging HIT T-15 beta-cells, in the presence of high glucose, was due, in part, to reduced expression of a key regulator of insulin enhancer-mediated expression, somatostatin transcription factor-1 (STF-1). In this study, we have examined whether the activity of the other essential transcription regulators of insulin gene expression, the RIPE3b1 and insulin control element (ICE) activators, were also influenced in these HIT T-15 cells. The results show that the binding and trans-activation functions of the RIPE3b1 activator are reduced in parallel with the loss in STF-1 and insulin gene expression. In contrast, the regulatory properties of the ICE activator are unaffected. Our studies indicate that insulin gene transcription is inhibited by glucose through a mechanism involving reduced expression of both the RIPE3b1 and STF-1 activators in HIT T-15 cells but is independent of the ICE activator.

Medical genetics: advances in brief: Regulation of insulin gene expression by the IDDM associated, insulin locus haplotype

Medical genetics: advances in brief: Regulation of insulin gene expression by the IDDM associated, insulin locus haplotype

The LIM Domain Homeobox Gene isl-1 Is a Positive Regulator of Islet Cell-specific Proglucagon Gene Transcription

The LIM domain homeobox gene islet 1 (isl-1) is expressed in the embryonic nervous system and may be an early marker of motor neuron specification. isl-1 is expressed in all 4 islet cell types, but a role for isl-1 in the regulation of insulin gene expression has not been demonstrated, and the genetic targets for isl-1 in the pancreas remain unknown. We show here that the proximal rat proglucagon gene promoter binds an amino-terminally truncated Trp-E-isl-1 fusion protein that lacks the LIM domains. The proglucagon gene promoter also binds full-length in vitro translated isl-1 containing the intact LIM domains. isl-1 antisera detects binding of proglucagon gene sequences to isl-1 present in a slowly-migrating complex in nuclear extracts from InR1-G9 islet cells. The transcriptional properties of the proglucagon gene promoter sequences that bind isl-1 (designated Ga, Gb, and Gc) were assessed after transfection of reporter genes into wild type and isl-1-antisense (isl-1(AS)) InR1-G9 islet cells. The proximal proglucagon gene (Ga) promoter sequence reduced TK-CAT activity by approximately 50%, but no change in the activity of the Ga-TK-CAT plasmid was seen after transfection of isl-1(AS) InR1-G9 cells. In contrast, the Gb/Gc sites activated transcription 2-3 fold in wild type InR1-G9 cells, and the isl-1-dependent activation of gene transcription through the Gb/Gc element was eliminated following transfection of isl-1(AS) InR1-G9 cells. These data demonstrate that the LIM domain homeobox gene isl-1 1) is not constrained from DNA binding by its LIM domains and 2) functions as a positive regulator of proglucagon gene transcription in the endocrine pancreas.

Functional analysis of a newly identified TAAT-box of the rat insulin-II gene promoter

Transcriptional regulation of insulin gene expression is achieved by an interplay of tissue-specific and ubiquitous cis- and trans-acting elements. E-box like motifs and TAAT-motifs were shown to play a crucial role in initiating insulin gene transcription. Studying the AT-rich region of the rat insulin-II promoter betwee nucleotides −212 and −196, we observed a base difference at −211, an adenosine instead of a cytidine, compared to the previously reported sequence (EMBL Accession No. J00748). Sequence analysis of promoter fragments from different rat strains showed that adenosine at position −211 represents the wild type (EMBL Accession No. X82162). This base exchange leads to the formation of an additional TAAT-motif, i.e. TAAT3, at the complementary DNA strand directly upstream of the previously studied TAAT2 motif, formerly named CT-2. Here we show that the newly identified motif TAAT3 is involved in (i) transcriptional control in vivo, (ii) in vitro DNA/protein interactions, and that (iii) TAAT1, TAAT2 and TAAT3 are binding sites for the homeodomain-containing factor IPF-1.

Regulation of insulin gene expression by the IDDM associated, insulin locus haplotype.

A 4.1 kb genomic region, spanning the insulin (INS) gene, confers genetic susceptibility to Type 1 or insulin-dependent diabetes mellitus (IDDM). Ten polymorphisms within this region form two predominant, complementary haplotypes. We have been studying the effects of these polymorphisms on the levels of insulin mRNA. Cloned genomic DNA fragments representing these two separate haplotypes were transiently transfected into a rodent pancreatic beta cell line, HIT-T15. These studies revealed that insulin mRNA levels were consistently higher in the transfectants expressing the diabetic haplotype. Over-expression of insulin mRNA may provide the basic mechanism for the diabetic susceptibility encoded at the INS locus.

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)

Localization of the genes encoding two transcription factors, LMX1 and CDX3, regulating insulin gene expression to human chromosomes 1 and 13.

The human lmx-1 and cdx-3 genomic clones {lambda}F11.1 and {lambda}F4.1, respectively, were isolated from a human genomic library by cross-hybridization with cDNA clones encoding Syrian hamster lmx-1 and cdx-3. The identities of the two clones were confirmed by DNA sequencing. The chromosomal locations of the human lmx-1 (LMX1) and cdx-3 (CDX3) genes were determined by fluorescence in situ hybridization. {lambda}F11.1 and {lambda}F4.1 DNAs were labeled with biotin by nick-translation in the presence of biotin-11-dUTP.

Insulin regulation of malic enzyme gene expression in rat liver: evidence for nuclear proteins that bind to two putative insulin response elements.

Diabetes in rats is characterized by insulin deficiency accompanied by a decrease in lipogenic enzymes. The malic enzyme (ME) gene, which encodes an important lipogenic enzyme, was used to investigate insulin regulation of gene expression. ME mRNA levels were reduced by more than 90% in the liver of diabetic rats. The administration of insulin (3 U/15 days) to either control or diabetic rats increased ME mRNA by 2- to 10-fold, respectively. Since diabetes reduces circulating T3 and the levels of nuclear T3-receptors, the potential role of thyroid hormone on insulin regulation of ME gene expression was also evaluated in thyroidectomized-diabetic rats. In these animals the levels of ME mRNA were undetectable but were increased by insulin even in the absence of thyroid hormones. These in vivo effects of insulin and T3 were not additive. The transcription rate of the gene was also reduced in the diabetic liver and recovered after insulin therapy. By computer analyses we have identified two different putative insulin response elements (IREs) in the ME gene promoter, hereafter referred to as IRE-I (-683 to -692), which is similar to the phosphoenol pyruvate carboxy kinase promoter IRE and IRE-II (-161 to -170), which is similar to the glyceraldehyde phosphate dehydrogenase gene promoter IRE-A. Results from gel retardation assays suggest that a single nuclear protein binds to IRE-I whereas two different nuclear proteins bind to IRE-II. The protein/IRE-I complex increased in liver nuclear extracts from diabetic rats and decreased after insulin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin stimulates phosphorylation of c-Jun, c-Fos, and Fos-related proteins in cultured adipocytes.

In differentiated 3T3-F442A adipocytes, insulin stimulated rapid and transient phosphorylation of c-Jun. Insulin also stimulated phosphorylation of c-Fos and several Fos-related proteins (pp72, pp45, and pp39) as indicated by precipitation with anti-c-Fos antibody following exposure to denaturating conditions. Phosphorylation of c-Fos was stimulated by 7-fold by 60 min, while phosphorylation of Fos-related proteins reached maxima of 3.5-5.5-fold at 15 to 60 min. The increase in phosphorylated c-Fos was due to an increase in both c-Fos protein and the stoichiometry of c-Fos phosphorylation, and was not observed in c-fos (-/-) cells. Additionally, insulin stimulated phosphorylation of a protein with molecular mass of approximately 82 kDa on tyrosine residues by 2.5-fold within 30 min; this protein appeared to be immunologically related to c-Fos. These increases in the phosphorylation of AP-1 transcription factors correlated with a > 5-fold stimulation of expression of a 12-O-tetradecanoylphorbol-13-acetate-responsive element-chloramphenicol acetyltransferase reporter gene transiently transfected into 3T3-F442A cells. These results indicate that insulin stimulates the phosphorylation of AP-1 transcription factors and several Fos-related proteins on serine and tyrosine residues. This is associated with changes in AP-1-mediated gene expression in vivo, suggesting that AP-1 phosphorylation by insulin plays a role in insulin-regulated gene expression.

Insulin gene transcription is decreased rapidly by lowering glucose concentrations in rat islet cells.

Glucose is the main physiological regulator of insulin biosynthesis and secretion. To exert an effect on biosynthesis, stimulatory concentrations of glucose act on insulin gene expression at both transcriptional and posttranscriptional levels. The relative importance of these two effects is controversial. Studies on the effect of decreasing glucose concentrations in vitro have been contradictory as to its rapidity and magnitude. We have investigated whether a decrease in glucose concentrations from a stimulatory (11 mM) to a nonstimulatory (2 mM) level regulates insulin gene expression in both isolated rat islets and INS-1 cells, an insulinoma-derived cell line that retains a number of the differentiation characteristics of native beta-cells. We show that, in the presence of a nonstimulatory concentration of glucose, insulin mRNA levels decrease rapidly in both rat islets and INS-1 cells. Low glucose decreases insulin gene expression at the transcriptional level, and this effect is mediated by DNA sequences present within 254 base pairs of the 5'-flank of the rat insulin I gene. We conclude that lowering glucose from 11 to 2 mM decreases insulin mRNA rapidly by a transcriptional effect.

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.

Nutrient regulation of insulin gene expression.

The beta cell of the islets of Langerhans contributes along with other factors to glucose homeostasis by sensing changes in the plasma glucose concentrations and adjusting the rate of insulin production and release. Over short periods of time, insulin production is controlled principally through translation of pre-existing mRNA. Over longer periods, insulin mRNA levels are modulated through effects on the rate of transcription of the insulin gene, and also through changes in the rate of decay of insulin mRNA. These long-term effects may be important in allowing the beta cell to adapt to changes in diet or periods of fasting. Several mechanisms involved in the control of the rate of translation of insulin mRNA have been described. Effects of glucose metabolism on the turnover of insulin mRNA have yet to be characterized in detail. At the level of transcription, cis-acting DNA elements and trans-acting factors involved in the transient response of the insulin gene to changes in intracellular cAMP levels, or to signals generated as a result of glucose metabolism, have been identified.

Protein Kinase Activity of the Insulin Receptor Is Essential for Insulin-Regulated Gene Expression

Protein Kinase Activity of the Insulin Receptor Is Essential for Insulin-Regulated Gene Expression

Protein Kinase Activity of the Insulin Receptor Is Essential for Insulin-Regulated Gene Expression.

Two Chinese hamster ovary (CHO) cell lines stably transfected with human insulin receptor cDNA, CHO-wt and CHO-mut, which express an equivalent number of normal and kinase-defective human insulin receptors, respectively, were used to assess the roles of insulin receptor tyrosine kinase activity in insulin-regulated gene expression. The effect of insulin on gene-33-promoter-driven chloramphenicol acetyltransferase (CAT), RSVLTR-driven beta-galactosidase (pRSVLTR-betagal) and SV40 late-promoter-driven hepatitis B surface antigen (pMLSV(2)HBsAg) were examined in CHO-wt and CHO-mut cells. Insulin-stimulated gene 33 promoter is 10- to 50-fold more effective in CHO-wt cells than that in parental CHO cells. However, no enhancement of insulin sensitivity of gene 33 promoter in CHO-mut cells relative to parental CHO cells was found. Similar phenomena were also observed, in that insulin regulated pRSVLTR-betagal and pMLSV(2)HBsAg in these three CHO lines. Our data indicated that the protein kinase activity of the insulin receptor is essential for the stimulatory activity of insulin toward the activities of different promoters. Copyright 1994 S. Karger AG, Basel

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.

Cyclic adenosine monophosphate prevents the glucocorticoid-mediated inhibition of insulin gene expression in rodent islet cells.

Dexamethasone negatively regulates insulin gene expression in HIT-15 cells. In vivo, however, an excess of glucocorticoids results in an increase in insulin biosynthesis and peripheral hyperinsulinemia. To resolve this contradiction, we have studied the effects of dexamethasone in primary rat islet cells. We show here that dexamethasone decreases insulin mRNA levels in single islet cells, as in HIT-15 cells, but does not affect these levels in reaggregated islet cells and increases them in intact islets of Langerhans. Because cAMP is an important regulator of insulin gene expression and intracellular cAMP content may be decreased in single beta cells, we investigated whether cAMP could prevent the inhibitory effect of dexamethasone on insulin mRNA levels. In the presence of cAMP analogues, the inhibitory action of dexamethasone was not only prevented, but insulin mRNA increased to levels comparable to those observed when cAMP analogues were used alone. We conclude that the insulin gene is negatively regulated by dexamethasone in single islet cells, but that other factors such as cAMP prevent this effect when the native environment of islet cells is preserved. Our results indicate that insulin gene regulation is influenced by cell to cell contacts within the islet, and that intracellular cAMP levels might be influential in this regulation.

1992 R.D. Lawrence Lecture. The regulation of insulin gene expression.

Diabetic MedicineVolume 9, Issue 9 p. 792-798 1992 R.D. Lawrence Lecture. The Regulation of Insulin Gene Expression K. Docherty, Corresponding Author K. Docherty Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, UKDepartment of Medicine, Queen Elizabeth Hospital, Birmingham, B15 2TH, UK.Search for more papers by this author K. Docherty, Corresponding Author K. Docherty Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, UKDepartment of Medicine, Queen Elizabeth Hospital, Birmingham, B15 2TH, UK.Search for more papers by this author First published: November 1992 https://doi.org/10.1111/j.1464-5491.1992.tb01896.xCitations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume9, Issue9November 1992Pages 792-798 RelatedInformation