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PubMed1999

Divergent regulation of human and rat proglucagon gene promoters in vivo.

A single mammalian proglucagon gene is expressed in the brain, islets, and intestinal enteroendocrine cells, which gives rise to a unique profile of proglucagon-derived peptides (PGDPs) in each tissue. The biological importance of glucagon, glucagon-like peptide (GLP)-1, and GLP-2 has engendered considerable interest in the factors regulating the synthesis and secretion of the PGDPs in vivo. Although rat proglucagon gene transcription has been extensively studied, the factors important for control of human proglucagon gene expression have not been examined. We now report that, despite conservation of proximal promoter G1-G4 enhancer-like elements, human proglucagon reporter plasmids containing these elements are transcriptionally inactive in islet cell lines. Remarkably, larger human proglucagon promoter fragments, such as the 1604 hGLU-Luc, are expressed in GLUTag enteroendocrine cells but not in islet cell lines. A total of 5775 bases of human proglucagon promoter were required for expression in islet cell lines. Analysis of human proglucagon promoter expression in transgenic mice demonstrated that approximately 1.6 kb of human proglucagon gene sequences directs expression of a human growth hormone reporter gene to the brain and intestinal enteroendocrine cells but not islet cells in vivo. These findings provide the first evidence demonstrating divergence in the mechanisms utilized for tissue-specific regulation of the human and rodent proglucagon genes.

Animals↗
PubMed2003

Impaired intestinal proglucagon processing in mice lacking prohormone convertase 1.

The neuroendocrine prohormone convertases 1 and 2 (PC1 and PC2) are expressed in endocrine intestinal L cells and pancreatic A cells, respectively, and colocalize with proglucagon in secretory granules. Mice lacking PC2 have multiple endocrinopathies and cannot process proglucagon to mature glucagon in the pancreas. Disruption of PC1 results in dwarfism and also multiple neuroendocrine peptide processing defects. This study compares the pancreatic and intestinal processing of proglucagon in mice lacking PC1 expression with that in age-matched wild-type controls. Because proglucagon was found to precipitate in acidic extracts, the intestinal processing profile was analyzed in both acidic and neutral extracts by gel filtration, HPLC, and RIA. Supporting a central role for PC2 in glucagon biosynthesis, we found normal processing of proglucagon to glucagon in the pancreas, whereas the intestinal proglucagon processing showed marked defects. Tissue proglucagon levels in null mice were elevated, and proglucagon processing to glicentin, oxyntomodulin, and glucagon-like peptide-1 and -2 (GLP-1 and GLP-2) was markedly decreased, indicating that PC1 is essential for the processing of all the intestinal proglucagon cleavage sites. This includes the monobasic site R(77) and, thereby, production of mature, biologically active GLP-1. We also found elevated glucagon levels, suggesting that factors other than PC1 that are capable of processing to mature glucagon are present in the secretory granules of the L cell. These findings strongly suggest that PC1 is essential for intestinal proglucagon processing in vivo and, thereby, plays an important role in production of the incretin hormone GLP-1 and the intestinotrophic hormone GLP-2.

Animals↗
PubMed1997

The caudal homeobox protein cdx-2/3 activates endogenous proglucagon gene expression in InR1-G9 islet cells.

The proglucagon gene is expressed in a highly cell-specific manner in islet and enteroendocrine cells. DNA sequences within the proximal proglucagon G1 promoter region bind the homeobox protein cdx-2/3, and cdx-2/3 activates the proglucagon promoter in fibroblasts. We show here that cdx-2/3 activates the proglucagon promoter in both islet (InR1-G9) and enteroendocrine (STC-1 and GLUTag) cell lines. Furthermore, transfected cdx-2/3 increased the levels of endogenous proglucagon mRNA transcripts in both transient and stable transfections of InR1-G9 islet cells. The cdx-2/3-dependent induction of endogenous proglucagon mRNA transcripts in stable islet lines was associated with a corresponding increase in the transcriptional activity of proglucagon promoter-luciferase plasmids. An amino-terminally truncated cdx-2/3 derivative containing the homeodomain and carboxy-terminal region of the molecule inhibited both the cdx-2/3 activation of the proglucagon promoter and the induction of endogenous proglucagon mRNA transcripts. These observations demonstrate that cdx-2/3, acting through the proximal G1 element, is a major transcriptional determinant of cell-specific proglucagon gene expression in pancreatic islet cells.

Animals↗
PubMed1995

Processing of mouse proglucagon by recombinant prohormone convertase 1 and immunopurified prohormone convertase 2 in vitro.

The mouse tumor cell line alpha TC1-6 was used as a model system to examine the post-translational processing of proglucagon. Determination of the mouse preproglucagon cDNA sequence and comparison with the published sequences of rat and human preproglucagons revealed nucleic acid homologies of 89.1 and 84%, respectively, and amino acid homologies of 94 and 89.4%, respectively. Immunohistochemical analyses with antibodies directed against PC2 and glucagon colocalized both the enzyme and substrate within the same secretory granules. PC1 was also immunolocalized in secretory granules. Cells were metabolically labeled with [3H]tryptophan, and extracts were analyzed by reverse-phase high pressure liquid chromatography. Radioactive peptides with retention times identical to those of synthetic peptide standards were recovered and subjected to peptide mapping to verify their identities. To determine the potential role of PC1 and PC2 in proglucagon processing, 3H-labeled proglucagon was incubated in vitro with recombinant PC1 and/or immunopurified PC2. Both enzymes cleaved proglucagon to yield the major proglucagon fragment, glicentin, and oxyntomodulin, whereas only PC1 released glucagon-like peptide-I from the major proglucagon fragment. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. These results suggest a potential role for PC1 and/or PC2 in cleaving several of the normal products, excluding glucagon, from the mouse proglucagon precursor.

Amino Acid Sequence↗
PubMed1997

Role of the prohormone convertase PC3 in the processing of proglucagon to glucagon-like peptide 1.

Proglucagon is processed differentially in pancreatic alpha-cells and intestinal endocrine L cells to release either glucagon or glucagon-like peptide-1-(7-36amide) (tGLP-1), two peptide hormones with opposing biological actions. Previous studies have demonstrated that the prohormone convertase PC2 is responsible for the processing of proglucagon to glucagon, and have suggested that the related endoprotease PC3 is involved in the formation of tGLP-1. To understand better the biosynthetic pathway of tGLP-1, proglucagon processing was studied in the mouse pituitary cell line AtT-20, a cell line that mimics the intestinal pathway of proglucagon processing and in the rat insulinoma cell line INS-1. In both of these cell lines, proglucagon was initially cleaved to glicentin and the major proglucagon fragment (MPGF) at the interdomain site Lys70-Arg71. In both cell lines, MPGF was cleaved successively at the monobasic site Arg77 and then at the dibasic site Arg109-Arg110, thus releasing tGLP-1, the cleavages being less extensive in INS-1 cells. Glicentin was completely processed to glucagon in INS-1 cells, but was partially converted to oxyntomodulin and very low levels of glucagon in AtT-20 cells in the face of generation of tGLP-1. Adenovirus-mediated co-expression of PC3 and proglucagon in GH4C1 cells (normally expressing no PC2 or PC3) resulted in the formation of tGLP-1, glicentin, and oxyntomodulin, but no glucagon. When expressed in alphaTC1-6 (transformed pancreatic alpha-cells) or in rat primary pancreatic alpha-cells in culture, PC3 converted MPGF to tGLP-1. Finally, GLP-1-(1-37) was cleaved to tGLP-1 in vitro by purified recombinant PC3. Taken together, these results indicate that PC3 has the same specificity as the convertase that is responsible for the processing of proglucagon to tGLP-1, glicentin and oxyntomodulin in the intestinal L cell, and it is concluded that this enzyme is thus able to act alone in this processing pathway.

Animals↗
PubMed2002

Transcriptional activation of the proglucagon gene by lithium and beta-catenin in intestinal endocrine L cells.

The proglucagon gene encodes several peptide hormones that regulate blood glucose homeostasis, growth of the small intestine, and satiety. Among them, glucagon-like peptide 1 (GLP-1) lowers blood glucose levels in patients with diabetes and inhibits eating and drinking in fasted rats. Although proglucagon transcription and GLP-1 synthesis were shown to be activated by forskolin and other protein kinase A (PKA) activators, deleting or mutating the cAMP-response element (CRE) only moderately attenuates the proglucagon gene promoter in response to PKA activation. Therefore, PKA may activate proglucagon transcription via a mechanism independent of the CRE motif. Recently, PKA was shown to phosphorylate and inactivate GSK-3beta, a key mediator in the Wnt signaling pathway. We show here that lithium, an inhibitor of GSK-3beta, activates proglucagon gene transcription and stimulates GLP-1 synthesis in an intestinal endocrine L cell line, GLUTag. The activation was also observed in primary fetal rat intestinal cell (FRIC) cultures, but not in a pancreatic A cell line. Co-transfection of beta-catenin, a downstream effector of GSK-3beta activities, activated the proglucagon gene promoter without a CRE. Furthermore, forskolin and 8-Br-cAMP phosphorylated GSK-3beta at serine 9 in intestinal proglucagon-producing cells, and both lithium and forskolin induced the accumulation of free beta-catenin in these cell lines. These observations indicate that the proglucagon gene is among the targets of the Wnt signaling pathway.

Cell Line↗
PubMed1999

Lamprey proglucagon and the origin of glucagon-like peptides.

We characterized two proglucagon cDNAs from the intestine of the sea lamprey Petromyzon marinus. As in other vertebrates, sea lamprey proglucagon genes encode three glucagon-like sequences, glucagon, and glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). This observation indicates that all three glucagon-like sequences encoded by the proglucagon gene originated prior to the divergence of jawed and jawless vertebrates. Estimates of the rates of evolution for the glucagon-like sequences suggest that glucagon originated first, about 1 billion years ago, while GLP-1 and GLP-2 diverged from each other about 700 MYA. The two sea lamprey intestinal proglucagon cDNAs have differing coding potential. Proglucagon I cDNA encodes the previously characterized glucagon and the glucagon-like peptide GLP-1, while proglucagon II cDNA encodes a predicted GLP-2 and, possibly, a glucagon. The existence of two proglucagon cDNAs which differ with regard to their potential to encode glucagon-like peptides suggests that the lamprey may use differential gene expression as a third mechanism, in addition to alternative proteolytic processing and mRNA splicing, to regulate the production of proglucagon-derived peptides.

Amino Acid Sequence↗
PubMed1997

POU domain transcription factor brain 4 confers pancreatic alpha-cell-specific expression of the proglucagon gene through interaction with a novel proximal promoter G1 element.

The proglucagon gene is expressed in a highly restricted tissue-specific manner in the alpha cells of the pancreatic islet, the hypothalamus, and the small and large intestines. Proglucagon is processed to glucagon and glucagon-like peptides GLP-1 and -2. Glucagon is expressed in alpha cells and regulates glucose homeostasis. GLP-1 is implicated in the control of insulin secretion, food intake, and satiety signaling, and GLP-2 is implicated in regulating small-bowel growth. Cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element which contains several AT-rich domains with binding sites for homeodomain transcription factors. In an attempt to identify major homeodomain proteins involved in pancreatic alpha-cell-specific proglucagon expression, we found that the POU domain transcription factor brain 4 is abundantly expressed in proglucagon-producing islet cell lines and rat pancreatic islets. In the latter, brain 4 and glucagon immunoreactivity colocalize in the outer mantle of islets. Electrophoretic mobility shift assays with specific antisera identify brain 4 as a major constituent of nuclear proteins of glucagon-producing cells that bind to the G1 element of the proglucagon gene proximal promoter. Transcriptional transactivation experiments reveal that brain 4 is a major regulator of proglucagon gene expression by its interaction with the G1 element. The finding that a neuronal transcription factor is involved in glucagon gene transcription may explain the presence of proglucagon in certain areas of the brain as well as in pancreatic alpha cells. Further, this finding supports the idea that the neuronal properties of endodermis-derived endocrine pancreatic cells may find their basis in regulation of gene expression by neuronal transcription factors.

Animals↗
PubMed2005

Intron 1 sequences are required for pancreatic expression of the human proglucagon gene.

The mammalian proglucagon gene is expressed in pancreatic islet A-cells, intestinal L-cells, and select neurons of the brain, where posttranslational processing results in the liberation of a unique profile of peptides. Despite the importance of proglucagon-derived peptides in human biology, little is known about the regulation of the human gene, as the rat gene has been the preferred model for understanding the regulation of proglucagon gene expression. Previously, we have shown that although the immediate promoter region of the rat proglucagon gene is sufficient for expression in pancreatic islet cells, the homologous human proglucagon promoter sequences are not sufficient. We have now used a comparative genomic approach to identify noncoding sequences near the human proglucagon gene that are conserved among mammals, and thus potentially are regulatory sequences. Our alignments identified three evolutionarily conserved noncoding regions (ECR), one is the immediate promoter region (ECR1), the second is about 5 kb 5' to the mRNA start site (ECR2), and the third is near the 3' end of the first intron (ECR3). Our in vitro transient transfection assays with reporter gene constructs that include the human ECR3 support expression in rodent islet cell lines. Complementary studies with transgenic mice possessing a reporter gene regulated by a human proglucagon gene promoter-intron 1 (including ECR3) sequences express the reporter gene in the pancreas, as well as the intestine and selected neurons. These studies suggest that conserved sequences within intron 1 of the human proglucagon gene are important for expression in the pancreas.

Animals↗
PubMed1998

Insulin, but not glucose lowering corrects the hyperglucagonemia and increased proglucagon messenger ribonucleic acid levels observed in insulinopenic diabetes.

The factors that regulate glucagon biosynthesis and proglucagon gene expression are poorly defined. We previously reported that insulin inhibits proglucagon gene expression in vitro. In vivo, however, the effects of insulin on the regulation of the proglucagon gene have been controversial. Furthermore, whether glucose plays any role alone or in conjunction with insulin on proglucagon gene expression is unknown. We investigated the consequences of insulinopenic diabetes on glucagon gene expression in the endocrine pancreas and intestine and whether insulin and/or glucose could correct the observed abnormalities. We show here that in the first 3 days after induction of hyperglycemia by streptozotocin, rats have levels of plasma glucagon and proglucagon messenger RNA comparable to those of normoglycemic controls despite hyperglycemia. With more prolonged diabetes, plasma glucagon and proglucagon messenger RNA levels increase; this increase is corrected by insulin treatment, but not by phloridzin despite normalization of the glycemia by both treatments. Proglucagon gene expression exhibits the same regulatory response to glucose and insulin in both pancreas and ileum. We conclude that insulin tonically inhibits proglucagon gene expression in the pancreas and ileum and that glucose plays a minor, if any, role in this regulation.

Animals↗
PubMed1996

Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: an antisense study.

To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, alpha TC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 +/- 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 +/- 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

Animals↗
PubMed1995

Trout and chicken proglucagon: alternative splicing generates mRNA transcripts encoding glucagon-like peptide 2.

In mammals, the proglucagon gene is transcribed into a single identical mRNA in pancreas, intestine, and brain. The proglucagon mRNA encodes glucagon and two glucagon-like peptides (GLP 1 and GLP 2), whose production is regulated by tissue-specific proteolytic processing. Previously characterized pancreatic proglucagon cDNAs from birds and fish encode glucagon and only one glucagon-like peptide, GLP 1. The isolation of intestinal proglucagon cDNAs from the rainbow trout, Oncorhynchus mykiss, and chicken, Gallus gallus, shows that the proglucagon gene of fish and birds also contains the sequence of a second glucagon-like peptide, GLP 2. In contrast to the proglucagon mRNAs from mammals, fish and bird proglucagon mRNAs from pancreas and intestine have different 3'-ends that are due to alternative mRNA splicing. The intestinal mRNA was found to be spliced to one or more exons, which encode GLP 2, while the pancreatic mRNA terminates within the intron between the exons encoding GLP 1 and GLP 2. These results show that proglucagon gene expression is regulated at the level of mRNA splicing and serve to reemphasize the potential biological importance of GLP 2.

Alternative Splicing↗
PubMed1986

Early O-glycosidic glycosylation of proglucagon in pancreatic islets: an unusual type of prohormonal modification.

Proglucagon from rat islets is identified as a glycoprotein by its binding to soybean lectin and by the biosynthetic incorporation of [14C]galactosamine. Glycosylation can be demonstrated for both forms of proglucagon, i.e. the primary translation product which is detectable as early as 30 s after incubation of isolated islets with radioactive amino acids (proglucagon a), and its conversion product of slightly higher electrophoretic mobility which is formed after 5-10 min of incubation (proglucagon b). This glycosylation is determined to be of the O-glycosidic type by the following criteria: rat proglucagon has previously been shown to lack an acceptor sequence for N-glycosidic linkage of sugars, the sugar bond in rat proglucagon is labile under mild alkaline conditions, glycosylated serine is demonstrated in proteolytic lysates of both the early and the late form of this prohormone. O-glycosidic linkage of sugars has not been reported for other prohormones. Its early formation and the apparent absence of N-glycosidically bound sugars in proglucagon give evidence for an unusual type of protein glycosylation.

Animals↗
PubMed2001

Identification of a proglucagon cDNA from Rana tigrina rugulosa that encodes two GLP-1s and that is alternatively spliced in a tissue-specific manner.

Glucagon plays a pivotal role in the regulation of metabolism. A glucagon receptor has been previously characterized in the frog, Rana tigrina rugulosa, and the frog and human glucagon receptors have been shown to possess similar binding affinities toward human glucagon. To study the structural evolution of glucagon peptide and its receptor in vertebrates, in the current study, a proglucagon cDNA from the same frog species was cloned. Interestingly, in contrast to the mammalian proglucagons that contain only one GLP-1 peptide, the frog proglucagon cDNA encodes two GLP-1 peptides (GLP-1A and GLP-1B) in addition to a glucagon peptide and a glucagon-like peptide 2 (GLP-2). By reverse transcriptase-PCR (RT-PCR) analysis, the proglucagon gene expression was widely detected in the brain, colon, small intestine, liver, lung, and pancreas, suggesting that the proglucagon-derived peptides have diverse functions in frogs. Moreover, tissue-specific alternative mRNA splicing was observed in the brain, colon, and pancreas. In these tissues, proglucagon transcripts with a 135 bp in frame deletion encoding GLP-1A were found. This splicing event in R. tigrina rugulosa is novel because it deletes a GLP-1 encoding sequence instead of the GLP-2 observed in other vertebrates. These findings should enhance understanding of the proglucagon evolution, structure, and expression in vertebrates.

Alternative Splicing↗
PubMed2006

Peptide YY and proglucagon mRNA expression patterns and regulation in the gut.

OBJECTIVE: Peptide YY (PYY) and glucagon-like peptide-1 are important in the control of energy homeostasis and are both secreted from the gut in response to ingested nutrients. However, more studies are needed on nutrient regulation of their gene expression patterns in specific areas of the gut. This study detailed PYY and proglucagon (the gene that encodes glucagon-like peptide-1) gene expression patterns and regulation in the gut. We further examined the regulation of PYY and proglucagon mRNA by a diet containing fermentation-resistant starch (in vivo) and butyrate (in vitro). RESEARCH METHODS AND PROCEDURES: Quantitative real time reverse transcriptase-polymerase chain reaction was used to measure PYY and proglucagon gene expression in epithelial cells collected from the duodenum, jejunum, cecum, and colon in normal Sprague-Dawley rats and in rats fed a resistant starch diet for 4 weeks. The same measurements were also performed in primary epithelial cells collected from the cecum and colon of normal rats after the cells were incubated with butyrate for 3 hours. RESULTS: The gene expression patterns for PYY and proglucagon are similar to their peptide distribution patterns in the gut. Also, PYY and proglucagon mRNA expression were up-regulated in the cecum and colon in resistant-starch-fed rats. Butyrate increased PYY and proglucagon gene expression in a dose-dependent manner in vitro. DISCUSSION: Our data provide evidence that the distal part of the gut has the ability to sense nutrients such as butyrate, resulting in the up-regulation of PYY and proglucagon gene expression.

Animals↗
PubMed1987

Primary structures of three fragments of proglucagon from the pancreatic islets of the daddy Sculpin (Cottus scorpius).

Three peptides isolated from the Brockmann bodies of the daddy sculpin, a teleostean fish, have been identified as fragments of one or more proglucagons. The peptide L Q D A E D S S R F D A D D T L A G E A R E L S T P K represents the NH2 terminus of proglucagon (residues 1-27), H S E G T F S N D Y S K Y L E T R R A Q D F V Q W L K N S represents glucagon and H A D G T F T S D V S S Y L N D Q A I K D F V A K L K S G K V represents the glucagon-like peptide at the COOH terminus of the precursor. The fast-atom bombardment mass spectra of the three peptides were consistent with the proposed structures and demonstrated that further posttranslational modifications of the peptides had not taken place. Sculpin glucagon is identical to anglerfish glucagon II but sculpin proglucagon(1-27) and glucagon-like peptide show stronger homology to the corresponding regions of anglerfish proglucagon I than to proglucagon II. The structures of the peptides are suggestive of the action of trypsin-like and carboxypeptidase-B-like enzymes at the site of pairs of basic amino acid residues in proglucagon. The presence of a COOH-terminal lysyl group in proglucagon(1-27) may indicate, however, that the penultimate prolyl residue partially inhibits the action of the carboxypeptidase-B-like activity.

Amino Acid Sequence↗
PubMed2003

Aberrant regulation of human intestinal proglucagon gene expression in the NCI-H716 cell line.

Despite interest in understanding glucagon-like peptide-1 (GLP-1) production, the factors important for GLP-1 biosynthesis remain poorly understood. We examined control of human proglucagon gene expression in NCI-H716 cells, a cell line that secretes GLP-1 in a regulated manner. Insulin, phorbol myristate acetate, or forskolin, known regulators of rodent proglucagon gene expression, had no effect, whereas sodium butyrate decreased levels of NCI-H716 proglucagon mRNA transcripts. The inhibitory effect of sodium butyrate was mimicked by trichostatin A but was not detected with sodium acetate or isobutyrate. The actions of butyrate were not diminished by the ERK1/2 inhibitor PD98059, p38 inhibitor SB203580, or soluble guanylate cyclase inhibitor LY83583 or following treatment of cells with KT5823, a selective inhibitor of cGMP-dependent protein kinase. NCI-H716 cells expressed multiple proglucagon gene transcription factors including isl-1, pax-6, pax-2, cdx-2/3, pax-4, hepatocyte nuclear factor (HNF)-3 alpha, HNF-3beta, HNF-3 gamma, and Nkx2.2. Nevertheless, the butyrate-dependent inhibition of proglucagon gene expression was not associated with coordinate changes in transcription factor expression and both the human and rat transfected proglucagon promoters were transcriptionally inactive in NCI-H716 cells. Hence, NCI-H716 cells may not be a physiologically optimal model for studies of human enteroendocrine proglucagon gene transcription.

Adenocarcinoma↗
PubMed2004

Pdx-1 is not sufficient for repression of proglucagon gene transcription in islet or enteroendocrine cells.

Pdx-1 plays a key role in the development of the pancreas and the control of islet gene transcription and has also been proposed as a dominant regulator of the alpha- vs. beta-cell phenotype via extinction of proglucagon expression. To ascertain the relationship between Pdx-1 and proglucagon gene expression, we examined the effect of enhanced pdx-1 expression on proglucagon gene expression in murine islet alphaTC-1 and GLUTag enteroendocrine cells. Although adenoviral transduction increased the levels of pdx-1 mRNA transcripts and nuclear Pdx-1 protein, overexpression of pdx-1 did not repress endogenous proglucagon gene expression in alphaTC-1 or GLUTag cells or murine islets. Immunohistochemical analysis of cells transduced with Ad-pdx-1 demonstrated multiple individual islet or enteroendocrine cells exhibiting both nuclear Pdx-1 and cytoplasmic glucagon-like peptide-1 immunopositivity. The failure of pdx-1 to inhibit endogenous proglucagon gene expression was not attributable to defects in Pdx-1 nuclear translocation or DNA binding as demonstrated using Western blotting and EMSA analyses. Furthermore, Ad-pdx-1 transduction did not repress proglucagon promoter activity in alphaTC-1 or GLUTag cells. Taken together, these findings demonstrate that pdx-1 alone is not sufficient for specification of the hormonal phenotype or extinction of proglucagon gene expression in islet or enteroendocrine cells.

Animals↗