Exendin-4 increases histone acetylase activity and reverses epigenetic modifications that silence Pdx1 in the intrauterine growth retarded rat.

AIMS/HYPOTHESIS: The abnormal intrauterine milieu of intrauterine growth retardation (IUGR) permanently alters gene expression and function of pancreatic beta cells leading to the development of diabetes in adulthood. Expression of the pancreatic homeobox transcription factor Pdx1 is permanently reduced in IUGR islets suggesting an epigenetic mechanism. Exendin-4 (Ex-4), a long-acting glucagon-like peptide-1 (GLP-1) analogue, given in the newborn period increases Pdx1 expression and prevents the development of diabetes in the IUGR rat. METHODS: IUGR was induced by bilateral uterine artery ligation in fetal life. Ex-4 was given on postnatal days 1-6 of life. Islets were isolated at 1 week and at 3-12 months. Histone modifications, PCAF, USF1 and DNA methyltransferase (Dnmt) 1 binding were assessed by chromatin immunoprecipitation (ChIP) assays and DNA methylation was quantified by pyrosequencing. RESULTS: Phosphorylation of USF1 was markedly increased in IUGR islets in Ex-4 treated animals. This resulted in increased USF1 and PCAF association at the proximal promoter of Pdx1, thereby increasing histone acetyl transferase (HAT) activity. Histone H3 acetylation and trimethylation of H3K4 were permanently increased, whereas Dnmt1 binding and subsequent DNA methylation were prevented at the proximal promoter of Pdx1 in IUGR islets. Normalisation of these epigenetic modifications reversed silencing of Pdx1 in islets of IUGR animals. CONCLUSIONS/INTERPRETATION: These studies demonstrate a novel mechanism whereby a short treatment course of Ex-4 in the newborn period permanently increases HAT activity by recruiting USF1 and PCAF to the proximal promoter of Pdx1 which restores chromatin structure at the Pdx1 promoter and prevents DNA methylation, thus preserving Pdx1 transcription.

Exendin-4 does not promote Beta-cell proliferation or survival during the early post-islet transplant period in mice.

Current pancreatic islet transplantation protocols achieve remarkable short-term success, but long-term insulin independence remains elusive. Hypoxic and inflammatory insults cause substantial early posttransplant graft loss while allo/autoimmunity and immunosuppressive drug toxicity threaten long-term graft mass and function. Exendin-4 (Ex4) is a GLP-1 receptor agonist that promotes beta-cell proliferation, survival, and differentiation. To determine whether Ex-4 displays potential as a graft-supportive agent, we transplanted 500 murine islets under the kidney capsule of syngeneic or allogeneic streptozocin-treated recipient mice and immediately initiated daily treatment with vehicle or Ex4. Graft beta-cell proliferation, death, and vascularity were assessed at 1, 3, and 10 days after syngeneic islet transplantation. For allogeneic recipients, blood glucose and body weight were assessed until glycemic deterioration. Ex-4 did not promote graft beta-cell proliferation, reduce beta-cell death, or enhance graft vascularity over the first 10 days after syngeneic islet transplantation. A trend toward prolongation of posttransplant euglycemia was observed with Ex4 treatment in nonimmune-suppressed allograft recipients, but its use in this setting was associated with frequent, severe hypoglycemia over the first 2 posttransplant days. Our findings do not support a beneficial effect of Ex-4 on islet grafts during the critical early posttransplant period, further, they demonstrate a significant hypoglycemic potential of Ex-4 in the first days after islet transplantation in mice. Optimal application of GLP-1 receptor agonists for long-term proliferative and survival benefits in transplantation may require earlier intervention prior to and/or during islet isolation for peri-transplant cytoprotection and administration beyond the period of engraftment.

Tissue-specific deletion of Foxa2 in pancreatic beta cells results in hyperinsulinemic hypoglycemia.

We have used conditional gene ablation to uncover a dramatic and unpredicted role for the winged-helix transcription factor Foxa2 (formerly HNF-3 beta) in pancreatic beta-cell differentiation and metabolism. Mice that lack Foxa2 specifically in beta cells (Foxa2(loxP/loxP); Ins.Cre mice) are severely hypoglycemic and show dysregulated insulin secretion in response to both glucose and amino acids. This inappropriate hypersecretion of insulin in the face of profound hypoglycemia mimics pathophysiological and molecular aspects of familial hyperinsulinism. We have identified the two subunits of the beta-cell ATP-sensitive K(+) channel (K(ATP)), the most frequently mutated genes linked to familial hyperinsulinism, as novel Foxa2 targets in islets. The Foxa2(loxP/loxP); Ins.Cre mice will serve as a unique model to investigate the regulation of insulin secretion by the beta cell and suggest the human FOXA2 as a candidate gene for familial hyperinsulinism.

Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas.

Diabetes is caused by a failure of the pancreas to produce insulin in amounts sufficient to meet the body's needs. A hallmark of diabetes is an absolute (type 1) or relative (type 2) reduction in the mass of pancreatic beta-cells that produce insulin. Mature beta-cells have a lifespan of approximately 48-56 days (rat) and are replaced by the replication of preexisting beta-cells and by the differentiation and proliferation of new beta-cells (neogenesis) derived from the pancreatic ducts. Here, we show that the insulinotropic hormone glucagon-like peptide (GLP)-1, which is produced by the intestine, enhances the pancreatic expression of the homeodomain transcription factor IDX-1 that is critical for pancreas development and the transcriptional regulation of the insulin gene. Concomitantly, GLP-1 administered to diabetic mice stimulates insulin secretion and effectively lowers their blood sugar levels. GLP-1 also enhances beta-cell neogenesis and islet size. Thus, in addition to stimulating insulin secretion, GLP-1 stimulates the expression of the transcription factor IDX-1 while stimulating beta-cell neogenesis and may thereby be an effective treatment for diabetes.

Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.

Diabetes is a disease of increasing prevalence in the general population and of unknown cause. Diabetes is manifested as hyperglycemia due to a relative deficiency of the production of insulin by the pancreatic beta-cells. One determinant in the development of diabetes is an inadequate mass of beta-cells, either absolute (type 1, juvenile diabetes) or relative (type 2, maturity-onset diabetes). Earlier, we reported that the intestinal hormone glucagon-like peptide I (GLP-I) effectively augments glucose-stimulated insulin secretion. Here we report that exendin-4, a long-acting GLP-I agonist, stimulates both the differentiation of beta-cells from ductal progenitor cells (neogenesis) and proliferation of beta-cells when administered to rats. In a partial pancreatectomy rat model of type 2 diabetes, the daily administration of exendin-4 for 10 days post-pancreatectomy attenuates the development of diabetes. We show that exendin-4 stimulates the regeneration of the pancreas and expansion of beta-cell mass by processes of both neogenesis and proliferation of beta-cells. Thus, GLP-I and analogs thereof hold promise as a novel therapy to stimulate beta-cell growth and differentiation when administered to diabetic individuals with reduced beta-cell mass.

Defective mutations in the insulin promoter factor-1 (IPF-1) gene in late-onset type 2 diabetes mellitus.

Type 2 diabetes mellitus is a common disabling disease with onset in middle-aged individuals, caused by an imbalance between insulin production and action. Genetic studies point to major genetic components, but, with the exception of maturity-onset diabetes of the young (MODY), specific diabetes susceptibility genes remain to be identified. Recent studies showed that a dominant negative mutation in the insulin promoter factor-1 (IPF-1), a pancreatic beta-cell specific transcription factor, causes pancreatic agenesis and MODY. Thus, we investigated 192 French, non-MODY type 2 diabetic families for mutations in IPF-1. We identified 3 novel IPF-1 mutations, including 2 substitutions (Q59L and D76N) and an in-frame proline insertion (InsCCG243). Functional transactivation assays of these IPF-1 mutant isoforms in a beta-pancreatic tumor cell line transfected with a transcriptional reporter and IPF-1 expression plasmids demonstrate a significant inhibition of basal insulin promoter activity (stronger with the InsCCG243 mutant). We find that the InsCCG243 mutation is linked, in 2 families, to an autosomal dominant-like late-onset form of type 2 diabetes, in which insulin secretion becomes progressively impaired. The lower penetrance D76N and Q59L mutations were more prevalent and were associated with a relative risk of 12.6 for diabetes and with decreased glucose-stimulated insulin-secretion in nondiabetic subjects. We propose that IPF-1 mutations can cause MODY or apparently monogenic late-onset diabetes and that they represent a significant risk factor for type 2 diabetes in humans.

Developmental expression of the homeodomain protein IDX-1 in mice transgenic for an IDX-1 promoter/lacZ transcriptional reporter.

Expression of the homeodomain transcription factor IDX-1 (also known as IPF-1, STF-1, and PDX-1) is required for pancreas development, because disruption of the gene in mice and humans results in pancreatic agenesis. During embryonic development the idx-1 gene is first expressed in a localized region of foregut endoderm from which the duodenum and pancreas later develop. To more fully understand the role of IDX-1 in pancreas development, transgenic mice expressing the Escherichia coli lacZ gene under control of the 5'-proximal 4.6 kb of the idx-1 promoter were created as a reporter for the developmental expression of IDX-1. Here we show that the determinants for the developmental and tissue-specific expression of the endogenous idx-1 gene are faithfully reproduced by the 4.6-kb region of the idx-1 promoter. Expression of lacZ is detected in the development of the exocrine and endocrine pancreas in pancreatic ducts, common bile and cystic ducts, pyloric glands of the distal stomach, Brunner's glands, the intestinal epithelium of the duodenum, and the spleen. The observed spatial and temporal pattern of lacZ expression directed by the IDX-1 promoter further supports an important role of IDX-1 in specifying the development of several endodermal structures within the midsegment of the body. An unexpected finding is that IDX-1 promoter-driven (transcriptional) lacZ activity does not always coincide with the localization of IDX-1 messenger RNA by in situ hybridization and IDX-1 protein by immunocytochemistry in adult rat duodenum, suggesting the existence of regulation of IDX-1 expression at the posttranscriptional level of expression of the idx-1 gene.

Insulin promoter factor-1 gene mutation linked to early-onset type 2 diabetes mellitus directs expression of a dominant negative isoprotein.

The homeodomain transcription factor insulin promoter factor-1 (IPF-1) is required for development of the pancreas and also mediates glucose-responsive stimulation of insulin gene transcription. Earlier we described a human subject with pancreatic agenesis attributable to homozygosity for a cytosine deletion in codon 63 of the IPF-1 gene (Pro63fsdelC). Pro63fsdelC resulted in the premature truncation of an IPF-1 protein which lacked the homeodomain required for DNA binding and nuclear localization. Subsequently, we linked the heterozygous state of this mutation with type 2 diabetes mellitus in the extended family of the pancreatic agenesis proband. In the course of expressing the mutant IPF-1 protein in eukaryotic cells, we detected a second IPF-1 isoform, recognized by COOH- but not NH2-terminal-specific antisera. This isoform localizes to the nucleus and retains DNA-binding functions. We provide evidence that internal translation initiating at an out-of-frame AUG accounts for the appearance of this protein. The reading frame crosses over to the wild-type IPF-1 reading frame at the site of the point deletion just carboxy proximal to the transactivation domain. Thus, the single mutated allele results in the translation of two IPF-1 isoproteins, one of which consists of the NH2-terminal transactivation domain and is sequestered in the cytoplasm and the second of which contains the COOH-terminal DNA-binding domain, but lacks the transactivation domain. Further, the COOH-terminal mutant IPF-1 isoform does not activate transcription and inhibits the transactivation functions of wild-type IPF-1. This circumstance suggests that the mechanism of diabetes in these individuals may be due not only to reduced gene dosage, but also to a dominant negative inhibition of transcription of the insulin gene and other beta cell-specific genes regulated by the mutant IPF-1.

Homeodomain protein IDX-1: a master regulator of pancreas development and insulin gene expression.

The homeodomain protein IDX-1 appears to be a "master regulator" of pancreas development and beta-cell differentiation and function. In murine gene inactivation models and in a human subject with a homozygous mutation of the IDX-1 gene, the pancreas fails to develop. In the adult endocrine pancreas, IDX-1 is primarily expressed in beta cells, where it is a key factor in the upregulation of insulin gene transcription and appears to have a role in the regulation of the somatostatin, glucokinase, glucose transporter-2, and islet amyloid polypeptide genes. Recent studies also suggest a role for IDX-1 in the neogenesis and proliferation of beta cells. The observed functions of IDX-1 and its downregulation in parallel with insulin in glucose-toxicity models implicate IDX-1 as a potential factor contributing to the pathogenesis of diabetes mellitus. Future directions include the use of conditional gene inactivation to determine more precisely the role of IDX-1 throughout endocrine pancreas differentiation and the exploration of IDX-1 as a potential target for gene therapy of diabetes mellitus.

Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM).

The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic domains necessary for the two-step formation of alpha-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA were identified by examining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contained exon A, the 315-nucleotide (nt) segment situated between the PHM and PAL domains and present in rPAM-1 but absent from rPAM-2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment encoding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane protein; rPAM-3a lacks a 204-nt segment including the transmembrane domain and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the PHM domain; alternative splicing generates a novel 3'-region encoding a COOH-terminal pentapeptide followed by 1.1 kb of 3'-untranslated region. The soluble rPAM-5 protein lacks PAL, transmembrane, and cytoplasmic domains. These three forms of PAM mRNA can be generated by alternative splicing. The major forms of PAM mRNA in both lobes of the pituitary are rPAM-3b and rPAM-2. Despite the fact that anterior and neurointermediate pituitary contain a similar distribution of forms of PAM mRNA, the distribution of PAM proteins in the two lobes of the pituitary is quite different. Although integral membrane proteins similar to rPAM-2 and rPAM-3b are major components of anterior pituitary granules, the PAM proteins in the neurointermediate lobe have undergone more extensive endoproteolytic processing, and a 75-kDa protein containing both PHM and PAL domains predominates. The bifunctional PAM precursor undergoes tissue-specific endoproteolytic cleavage reminiscent of the processing of prohormones.

Proadrenocorticotropin/endorphin production and messenger ribonucleic acid levels in primary intermediate pituitary cultures: effects of serum, isoproterenol, and dibutyryl adenosine 3',5'-monophosphate.

Long term primary rat intermediate pituitary cells cultured in complete serum-free medium (CSFM) were previously shown to exhibit a low basal secretory rate and maintain a stable cellular level of immunoactive pro-ACTH/endorphin (PAE)-related peptides. The present studies used biosynthetic labeling techniques to demonstrate that the biosynthesis of PAE-derived peptides declined as a function of the time the cultures were maintained in CSFM. Compared to hormone biosynthesis in freshly isolated cells, the ability of melanotropes maintained in CSFM to synthesize hormone declined approximately 3-fold after 5 days and 7-fold after 10 days. The endoproteolytic processing of PAE was not changed. This diminution in biosynthetic rate was not observed in serum-supplemented cultures; the rates of hormone secretion and hormone biosynthesis were both substantially higher in cultures maintained in serum-containing medium, suggesting that stimulatory factors were present in serum supplements. Chronic treatment of intermediate pituitary cultures in CSFM with 100 microM (Bu)2cAMP or 100 nM isoproterenol mimicked the effects of serum on total PAE production, hormone biosynthesis, and PAE mRNA levels. The diminished biosynthetic ability of cultures maintained in CSFM for several days could be restored by subsequent chronic (B mu)2cAMP or isoproterenol treatment. Taken together with previous information, these results suggest that melanotropes maintained in CSFM exist in a functionally basal state and that stimulatory agents, in addition to inhibitory inputs such as dopamine, exert significant regulatory control over intermediate pituitary lobe function in vivo.

Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C.

We have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the alpha subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to Gs, Gi, and Go, respectively. A single beta subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the alpha subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive Gs alpha relative to Gi alpha and Go alpha when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. alpha subunits of G-proteins were not detected in cilia detached from the nonchemosensory respiratory epithelium of the palate. Immunohistochemical studies using an antiserum against the beta subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the alpha subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. Prolonged incubation periods with these antisera tended to obliterate these differences in staining patterns, giving rise to staining by both antisera of the ciliary surface, the olfactory receptor cell membranes, the axon bundles, and the acinar cells of the glands. In addition to G-proteins, we have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding.(ABSTRACT TRUNCATED AT 250 WORDS)