Insulin Response Sequence Research Articles

Hepatic nuclear factor 3 is an accessory factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

Transcription of the hepatic phosphoenolpyruvate carboxykinase gene is stimulated by glucocorticoids and inhibited by insulin. The glucocorticoid response is mediated by a complex glucocorticoid response unit that consists of two glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and two accessory factor-binding sites (AF1 and AF2). The complete unit is required for the full glucocorticoid response. The dominant insulin effect is mediated in part through an insulin response sequence that is coincident with the AF2 element. Members of the hepatic nuclear factor 3 (HNF3) and CCAAT enhancer binding protein (C/EBP) families bind to the AF2 element; however, there is no correlation between binding of these factors and the ability of the AF2 element to mediate an insulin response. We show here that binding of HNF3 does correlate with the stimulation of the glucocorticoid response by the AF2 element and that C/EBP is apparently not involved in this effect. This requirement for HNF3 is quite specific since the substitution of elements known to enhance the action of the GR in other promoters fails to recapitulate AF2 accessory factor activity. By contrast, an HNF3-binding site from the transthyretin gene is able to substitute for the wild type AF2 sequence and elicit a maximal glucocorticoid response. Based on current and previous observations, the glucocorticoid response unit consists of four DNA elements that bind four different proteins. These are: AF1 (hepatic nuclear factor 4/chicken ovalbumin upstream promoter transcription factor), AF2 (HNF3), GR1 (GR), and GR2 (GR).

Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus.

Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed.

Upstream Stimulatory Factors Bind to Insulin Response Sequence of the Fatty Acid Synthase Promoter USF1 IS REGULATED

Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. The concentration or activity of FAS in liver and adipose tissue changes dramatically when animals are subjected to nutritional and hormonal manipulations. We previously reported that due to changes in transcription, FAS synthesis declines and increases in an insulin-dependent manner during fasting and refeeding, respectively, and that insulin administration of streptozotocin-diabetic mice stimulates FAS transcription. We previously mapped the FAS insulin response sequence (IRS) to the proximal promoter region from position -71 to position -50, which contains an E-box DNA binding motif. Here, using competition gel shift assays and specific upstream stimulatory factor (USF) antibodies, we identified USF1 and USF2 as major components of complexes that bind to the FAS IRS. UV-cross-linking experiments further supported that USFs bind the FAS IRS. We also found that the amount of the 43-kDa USF1 was dramatically increased in liver of refed rats. In contrast, the amount of USF2 remained the same in liver of fasted or refed rats. Moreover, a 17-kDa protein in both fasted and refed rat liver was recognized by anti-USF1 antibodies, and this 17-kDa USF1-related protein was expressed in a manner opposite to that of the 43-kDa USF1, i.e. high in liver of fasted rats and decreased in liver of refed rats. These data suggest that the regulation of USF expression may play an important role in the regulation of FAS transcription.

Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes.

The rate of transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein 1 (IGFBP-1) genes is stimulated by glucocorticoids and inhibited by insulin. In both cases, the effect of insulin is dominant, since it suppresses both basal and glucocorticoid-stimulated PEPCK or IGFBP-1 gene transcription. Analyses of both promoters by transfection of PEPCK or IGFBP-1-chloramphenicol acetyltransferase fusion genes into rat hepatoma cells has led to the identification of insulin response sequences (IRSs) in both genes. The core IRS, T(G/A)TTTTG, is the same in both genes, but the PEPCK promoter has a single copy of this element whereas the IGFBP-1 promoter has two copies arranged as an inverted palindrome. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of hepatic nuclear factor 3 (HNF-3), although the latter does so with a sixfold-lower relative affinity. Both the PEPCK and the IGFBP-1 IRSs also function as accessory factor binding sites required for the full induction of gene transcription by glucocorticoids. A combination of transient transfection and DNA binding studies suggests that HNF-3 is the accessory factor that supports glucocorticoid-induced gene transcription. In both genes, the HNF-3 binding site overlaps the IRS core motif(s). A model in which insulin is postulated to mediate its negative effect on glucocorticoid-induced PEPCK and IGFBP-1 gene transcription indirectly by inhibiting HNF-3 action is proposed.

A novel DNA/protein complex interacts with the insulin-like growth factor binding protein-1 (IGFBP-1) insulin response sequence and is required for maximal effects of insulin and glucocorticoids on promoter function

Glucocorticoids stimulate and insulin inhibits hepatic production of IGFBP-1 at the level of gene transcription. We previously identified contiguous insulin and glucocorticoid response sequences in the proximal rat IGFBP-1 promoter. This insulin response sequence (IRS) is palindromic ( CAAAACAAA TTATTTTG) and each half resembles an IRS in the phosphoenolpyruvate carboxykinase (PEPCK) gene. We have reported that both the IGFBP-1 and PEPCK IRSs bind hepatocyte nuclear factor-3 (HNF-3) proteins [1]. We now report that IRSs from the IGFBP-1 and PEPCK, as well as an IRS which also binds HNF-3 in the rat tyrosine aminotransferase (TAT) gene, also interact with another DNA/protein complex in gel shift studies. Further, methylation interferences studies, gel shift and transient transfection studies with site-specific mutations identified a single base in the first half of the IRS that is critical both for interactions with proteins in this complex, and for maximal effects of insulin and glucocorticoids, on promoter function. Of note, a 250-fold excess of an oligo containing a C/EBP binding site (but not other AT-rich sequences) inhibits the formation of this complex in gel shift assays. Nevertheless, interactions with this C/EBP site are negligible at lower titers (≤ 100-fold excess), and antibodies against known C/EBP proteins do not react with this complex. Similarly, preincubation with CHOP, a truncated member of the C/EBP family which contains a β-leucine zipper domain, does not prevent or alter the mobility of this novel DNA/protein complex, indicating that components of this complex do not form heterodimers with β-ZIP proteins. We conclude that HNF-3 proteins and this novel C/EBP-related DNA/protein complex may play an important role in mediating interactions between glucocorticoids and insulin in the regulation of IGFBP-1 and perhaps multiple hepatic genes.

Potential convergence of insulin and cAMP signal transduction systems at the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter through CCAAT/enhancer binding protein (C/EBP).

Adenosine 3',5'-monophosphate (cAMP) stimulates phosphoenolpyruvate carboxykinase (PEPCK) gene transcription, whereas insulin has the opposite effect. In H4IIE cells, the effect of insulin is dominant since it represses cAMP-stimulated transcription. Discrete cis-acting elements in the PEPCK promoter that serve as an insulin response sequence (IRS) and cAMP response element (CRE) have been identified. Here we show that common proteins can bind both elements, since: (i) an almost identical pattern of protein binding is seen when oligonucleotides representing either the IRS or the CRE are used as the labeled probe in a gel retardation assay and (ii) the unlabeled wild-type, but not mutated, CRE oligonucleotide competes for protein binding to the labeled IRS probe, and vice versa. Six homo- and heterodimer complexes interact with these DNA elements; the complexes are composed of three individual protein species: (a) 42-kDa C/EBP alpha, (b) 30-kDa C/EBP alpha, and (c) an unidentified 20-kDa factor termed p20- CRE/IRS Binding Protein (p20-C/IBP). These proteins have a 30-fold greater affinity for the CRE at room temperature, a difference explained by the rapid dissociation rate of protein bound to the IRS, since the association rate of protein binding to both the IRS and CRE is the same. Protease digestion experiments suggest that the proteins bind to the CRE and IRS in different conformations. The IRS and CRE both function in the context of a heterologous promoter to mediate effects of insulin and cAMP, respectively, but, although the PEPCK IRS and CRE bind common proteins, the PEPCK CRE is not a functional IRS and the PEPCK IRS is not a functional CRE.

Hepatocyte Nuclear Factor-3 (HNF-3) Binds to the Insulin Response Sequence in the IGF Binding Protein-1 (IGFBP-1) Promoter and Enhances Promoter Function

IGF binding protein-1 is an important short-term modulator of IGF bioavailability. Hepatic transcription of IGFBP-1 is increased by glucocorticoids and suppressed by insulin. We previously identified adjacent glucocorticoid and insulin response sequences ∼90 bp 5′ to the RNA cap site in the IGFBP-1 promoter. This insulin response sequence contains a sequence highly related (10/12 bases) to a consensus HNF-3 binding sequence. Gel shift and supershift studies confirm that this sequence binds HNF-3 α, β and γ. Co-expression of HNF-3β enhances IGFBP-1 promoter activity in NIH-3T3 cells. Mutation of this HNF-3 binding sequence disrupts this effect as well as the ability of glucocorticoids to stimulate and of insulin to inhibit IGFBP-1 promoter activity in H4IIE and HepG2 hepatoma cells. HNF-3 binding at this site may play an important role in the multihormonal regulation of hepatic IGFBP-1 gene expression.

The cyclic adenosine 3',5'-monophosphate- and the glucocorticoid-dependent enhancers are targets for insulin repression of tyrosine aminotransferase gene transcription.

The pathway of gluconeogenesis is activated in liver shortly after birth and is controlled by glucagon and glucocorticoids, which stimulate, and insulin, which inhibits, the expression of genes coding for gluconeogenic enzymes. To understand the molecular basis of this cell type-specific and coordinate control, we analyzed the cis-regulatory elements of the tyrosine aminotransferase gene, which confer liver cell-specific expression in dependence of these hormones. The cAMP-responsive element (CRE) of the TAT gene is an essential element within a liver-specific enhancer and is recognized by the CRE-binding protein (CREB) in a phosphorylation-dependent manner. The glucocorticoid response is mediated by a complex regulatory unit comprised of the glucocorticoid receptor and other transcription factor-binding sites. Here, we show that both the cAMP- and glucocorticoid-inducible enhancers are targets for the antagonistic effects of insulin. The insulin-responsive sequences coincide with the CREB-binding site of the cAMP-responsive enhancer and a hepatocyte nuclear factor-3-binding site within the glucocorticoid-responsive unit. This design of the hormone-dependent enhancers reflects the molecular mechanism underlying the onset of tyrosine aminotransferase expression at birth when insulin levels decrease and concentrations of glucagon and glucocorticoids increase.

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter

Identification of an insulin response element in the fatty acid synthase promoter.

We have previously reported that insulin increases fatty acid synthase (FAS) gene transcription, and that sequences responsible for positive regulation are located within the first 332 base pairs of the FAS promoter. To define minimal sequences required for insulin regulation within this region, chimeric constructs containing serial 5' deletions starting at -318 and extending through position +67 of the rat FAS gene ligated to the luciferase reporter gene were transfected into 3T3-L1 adipocytes. Insulin treatment at 10 nM increased luciferase activity 2-3-fold in 3T3-L1 adipocytes transfected with constructs containing progressive deletions from -318 to -67. This stimulation of the FAS promoter activity by insulin was dose-dependent. However, no effect of insulin was observed when fusion constructs containing FAS promoter sequences spanning from -25 or from -19 to +67 were transfected into adipocytes. These results suggest that the insulin response sequences of the FAS gene may be located in the region from -67 to -25. DNase I footprinting using liver nuclear extracts revealed a protected region spanning -71 and -50 in addition to a region near the putative TATA box. Gel mobility shift assays using the sequence from -71 to -50 as a probe revealed nuclear factor(s) from mouse liver and 3T3-L1 adipocytes that specifically complexed with this sequence. Mutational analysis of this region showed that sequences between -68 and -60 are essential for recognition and interaction with a trans-acting factor(s). Moreover, when three tandem repeats of the sequences spanning -68 to -52 were linked to the SV40 promoter and used for transfection, luciferase activity increased 3.6-fold in response to insulin treatment. Thus, we have identified novel cis-acting DNA sequences responsible for insulin regulation of the FAS gene, which interact with nuclear protein(s) from liver and adipocytes and which are found to share limited homology to insulin response sequences present in other genes.

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter.

Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced by the liver and regulated by glucocorticoids and insulin at the level of gene transcription. To identify DNA sequences mediating the effects of glucocorticoids and insulin on IGFBP-1 promoter activity we created luciferase reporter gene constructs and performed transfection studies in H4IIE hepatoma cells. Initial studies confirmed that the IGFBP-1 promoter is functional when inserted in the correct orientation, but not in the reverse orientation. Dexamethasone (DEX) increased promoter activity 10-fold, and insulin reversed this effect of DEX by 85% at 8 h. The effects of DEX were abolished when constructs were truncated to 89 bases from the RNA cap site, and DNase footprinting with the DNA-binding domain of the human glucocorticoid receptor identified an imperfect palindrome containing two receptor-binding sites separated by three nucleotides typical of a glucocorticoid response element (GRE) at this location. Mutation of either binding site (or half-site) disrupted the effects of DEX, confirming that this sequence functions as a GRE. Two other regions of the promoter also footprinted with the glucocorticoid receptor protein and contained sequences consistent with glucocorticoid receptor-binding sites; however, neither of these footprints contained the full structure expected of a functional GRE, and neither mutation nor deletion of these other sequences altered the effects of DEX on promoter activity. To identify the DNA sequences required for the effects of insulin on glucocorticoid-stimulated promoter activity, we created internal deletions throughout the IGFBP-1 promoter region. Deletion of the 22-basepair (bp) sequence immediately 5' from the GRE disrupted the effect of insulin and appeared to increase basal promoter activity at least 2-fold in each of eight experiments (P < 0.001 vs. intact promoter). This region of the IGFBP-1 promoter contains a 19-bp palindrome (CAAAACAAACTTATTTTG) that overlaps the 5'-end of the GRE and is fully conserved in the human IGFBP-1 promoter. Each half of this palindrome resembles previously identified insulin response sequences, and deletion/mutation analysis suggests that each half may contribute to the effects of insulin on promoter activity. Gel shift studies confirmed that this palindrome binds H4IIE nuclear proteins. In summary, we have identified a GRE in the 5'-promoter region of the rat IGFBP-1 gene approximately 90 bp up-stream from the RNA cap site as well as a contiguous 22-bp region that plays a critical role in mediating the effects of insulin on glucocorticoid-stimulated promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Impaired insulin-mediated up-regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression by ethanol exposure in vitro

Ethanol exposure causes hepatocellular injury, hepatic insufficiency, and impaired hepatocellular regenerative capacity. In the present study, we examined insulin-responsive cell growth, DNA synthesis, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in FOCUS hepatocellular carcinoma cells treated with 0 mM, 40 mM or 100 mM ethanol since, the GAPDH gene contains 5′ insulin responsive regulatory sequences, and insulin is required for liver regeneration. Exposure of FOCUS hepatocellular carcinoma cells to ethanol resulted in an immediate reduction in GAPDH mRNA levels, and it abolished the sharp increase in GAPDH protein content observed at 24 and 48 h in control cultures. Diminished responsiveness of GAPDH gene expression to insulin stimulation could represent an important mechanism by which ethanol intoxication impairs liver regeneration.

Insulin and phorbol esters act through the same DNA element to inhibit phosphoenolpyruvate carboxykinase gene transcription.

Conference Article| August 01 1992 Insulin and phorbol esters act through the same DNA element to inhibit phosphoenolpyruvate carboxykinase gene transcription R. M. O'Brien; R. M. O'Brien 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232-0615, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar P. C. Lucas; P. C. Lucas 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232-0615, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar T. Yamasaki; T. Yamasaki 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232-0615, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar M. T. Bonovich; M. T. Bonovich 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232-0615, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar C. D. Forest; C. D. Forest 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232-0615, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar D. K. Granner D. K. Granner 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232-0615, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1992) 20 (3): 686–690. https://doi.org/10.1042/bst0200686 Article history Received: April 09 1992 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation R. M. O'Brien, P. C. Lucas, T. Yamasaki, M. T. Bonovich, C. D. Forest, D. K. Granner; Insulin and phorbol esters act through the same DNA element to inhibit phosphoenolpyruvate carboxykinase gene transcription. Biochem Soc Trans 1 August 1992; 20 (3): 686–690. doi: https://doi.org/10.1042/bst0200686 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: AF, accessory factor binding site, CAT, chloramphenicol acetyltransferase, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GR, glucocorticoid receptor binding site, GRU, glucocorticoid-response unit, IRE, insulin response element, IRS, insulin-response sequence, neo, neomycin resistance gene, PEPCK, phosphoenolpyruvate carboxykinase, TK, thymidine kinase This content is only available as a PDF. © 1992 Biochemical Society1992 Article PDF first page preview Close Modal You do not currently have access to this content.

Regulation of Phosphoenolpyruvate Carboxykinase Gene Expression by Insulin. Use of the Stable Transfection Approach to Locate an Insulin Responsive Sequence

H4IIE rat hepatoma cells were stably transfected with various phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase (PEPCK-CAT) expression vectors. The regulation of the transfected genes was qualitatively similar to that of the endogenous PEPCK gene. CAT expression was increased in response to cAMP and dexamethasone and insulin overrode these effects at concentrations known to be effective in suppressing transcription of the endogenous gene. The effect of insulin was dominant, as it is with the endogenous gene. A series of 5',3', and internal deletions of the PEPCK gene promoter were used to show that this insulin response requires at least two separate elements. One insulin-responsive sequence is located between -468 and -402, relative to the transcription initiation site. The other is between -271 and +69.

Identification of a Sequence in the PEPCK Gene That Mediates a Negative Effect of Insulin on Transcription

Phosphoenolpyruvate carboxykinase (PEPCK) governs the rate-limiting step in gluconeogenesis. Glucocorticoids and adenosine 3',5'-monophosphate (cAMP) increase PEPCK gene transcription and gluconeogenesis, whereas insulin has the opposite effect. Insulin is dominant, since it prevents cAMP and glucocorticoid-stimulated transcription. Glucocorticoid and cAMP response elements have been located in the PEPCK gene and now a 15-base pair insulin-responsive sequence (IRS) is described. Evidence for a binding activity that recognizes this sequence is presented.

PEPCK Gene as Model of Inhibitory Effects of Insulin on Gene Transcription

Regulation of gene transcription is a major action of insulin. Most of the greater than 20 examples of this effect involve the stimulation of transcription, but a few involve an inhibition. The inhibition of transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene has been studied in detail. Most of this effect is exerted at the level of transcription initiation. Hormone effects on transcription are thought to be mediated through cis-acting DNA sequences located in the 5'-flanking sequence adjacent to the transcription initiation site. The techniques of transient and stable transfection of fusion genes containing various segments of the PEPCK-gene promoter are being used to locate the insulin-responsive sequences.

Independent regulation of nonallelic pancreatic amylase genes in diabetic mice.

Administration of streptozotocin produces a diabetic condition in mice characterized by a specific decrease in amylase synthesis in the pancreas as well as a substantial reduction in amylase mRNA concentration. We have studied this effect in mice of the congenic strains C3H.AmyYBR and C3H.AmyCE with multiple active copies of the pancreatic amylase structural gene. When mice of these strains are treated with streptozotocin, the magnitude of reduction in the synthesis of each amylase isozyme is different. These differences are reflected in the relative activities of isozyme-specific mRNAs in an in vitro translation assay. Administration of insulin results in partial restoration of normal phenotypes. The results provide genetic evidence that individual copies of the amylase structural gene are associated with divergent cis-acting insulin-responsive sequences.

Effect of temperature on coupling of insulin receptors to stimulation of glucose transport in isolated rat adipocytes

The ability of temperature to influence insulin binding, hormone stimulation of glucose transport, and the coupling of these two events was studied in isolated rat adipocytes. Insulin binding was reduced as the temperature rose from 16°C to 37°C. Both total and cell surface binding varied in an inverse linear manner with temperature, and Scatchard analysis suggested that this was caused by an altered affinity with no change in receptor number. The insulin responsiveness of 3-O-methyl-glucose transport increased with temperature from a four-fold stimulation at 16°C to 12-fold at 37°C. Insulin sensitivity was also higher at elevated temperatures with the EC 50 at 37°C (0.3 ng/mL) one-third that at 16°C (0.9 ng/mL). The stimulation by a single insulin concentration (0.75 ng/mL) varied linearly with temperature. Since the amount of cell-associated insulin that was internalized (non-acid extractable) also increased in a linear fashion with temperature, it was possible to compare the amount of insulin binding to the cell surface needed to attain a certain effect. At 37°C half-maximal stimulation was seen when each cell bound 1255 insulin molecules. The same level of effect required 2510 molecules per cell at 24°C and 6024 molecules per cell at 16°C. These results reveal several things about the insulin-response sequence in fat cells: (1) both total cell and cell surface binding vary inversely with temperature; (2) both insulin responsiveness and sensitivity increase with temperature; (3) the opposing behaviors of binding and sensitivity result in an increase in the effect of a set amount of insulin bound (the coupling efficiency); and (4) the coupling process between insulin receptors and glucose transport is regulated by temperature and could be subject to control by other physiologic factors.