The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Daniel J Drucker - One of the best experts on this subject based on the ideXlab platform.
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CLINICAL CASE SEMINAR Prolonged Gastrointestinal Transit in a Patient with a Glucagon-Like Peptide (GLP)-1- and-2-Producing Neuroendocrine Tumor
2016Co-Authors: Patricia L. Brubaker, Daniel J Drucker, Sylvia L Asa, Mark Redston, Carol Swallow, R. GreenbergAbstract:Neuroendocrine tumors overexpressing the Proglucagon-derived peptides have been associated with severe constipa-tion. The relationship between two of the intestinal proglu-cagon-derived peptides, glucagon-like peptide (GLP)-1 and-2, and delayed gastrointestinal transit, was characterized in a patient with a neuroendocrine Proglucagon-derived peptide tumor. A 60-yr-old female presented with intractable con-stipation and intermittent vomiting. Gastric, oral-ileal and colonic transit times, and plasma hormone levels were deter-mined before tumor resection. Expression of the Proglucagon-derived peptides by the tumor was determined by immuno-histochemistry, Northern blot analysis, HPLC, and RIA. Oral-cecal transit was more than 3 h, and a barium follow-through study showed dilated and thickened folds with most of the barium concentrated in the ileum at 24 h; residual bar-ium was identified in the colon at 14 d post ingestion. Circu-lating levels of GLP-1 and-2 were 300- to 400-fold elevated compared with levels in normal human subjects. Normal bowel function was restored by tumor resection. Consistent with the elevated plasma hormone levels, the tumor was found to express the Proglucagon gene, and immunoreactive GLP-1 and-2 were detected by both immunohistochemistry and RIA. Overexpression of glucagon-like peptide-1 and-2 is associated with markedly prolonged gastrointestinal transit in humans. These findings are consistent with a role for these peptides in the regulation of gastrointestinal motility. (J Clin Endocrinol Metab 87: 3078–3083, 2002) THE ILEAL BRAKE is a neurohormonal feedback mech-anism that delays gastric and intestinal transit time, thereby enhancing nutrient digestion and absorption in the proximal small intestine and preventing nutrient overflow into the distal gut (1). A number of gut peptides have been identified as possible effectors of intestinal motility (2), in
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Pdx-1 is not sufficient for repression of Proglucagon gene transcription in islet or enteroendocrine cells.
Endocrinology, 2004Co-Authors: Grace Flock, Xiemin Cao, Daniel J DruckerAbstract: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 α- vs. β-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 αTC-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 αTC-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 ...
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aberrant regulation of human intestinal Proglucagon gene expression in the nci h716 cell line
Endocrinology, 2003Co-Authors: Xiemin Cao, Grace Flock, David M Irwin, Caroline Choi, Daniel J DruckerAbstract: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)-3α, HN...
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Pax-6 Activates Endogenous Proglucagon Gene Expression in the Rodent Gastrointestinal Epithelium
Diabetes, 2003Co-Authors: Denny K.y. Trinh, Patricia L. Brubaker, Kai Zhang, Moazzem Hossain, Daniel J DruckerAbstract:The Proglucagon gene encodes pancreatic glucagon and the glucagon-like peptides, which exert diverse effects on nutrient absorption and assimilation. The therapeutic potential of glucagon-like peptide-1 (GLP-1) has fostered interest in development of cellular engineering approaches to augment endogenous intestinal-derived GLP-1 for the treatment of type 2 diabetes. We have used adenovirus technology to examine the potential roles of the transcription factors Cdx-2/3 and Pax-6 as activators of endogenous Proglucagon gene expression in enteroendocrine cell lines and in nontransformed rat intestinal cells. Adenoviral-expressed Cdx-2/3 and Pax-6 activated Proglucagon promoter-luciferase activity in baby hamster kidney (BHK) fibroblasts, HEK 293 cells, and enteroendocrine cell lines. Pax-6, but not Cdx-2/3, induced expression of the endogenous Proglucagon gene in enteroendocrine cell lines, but not in heterologous fibroblasts. Furthermore, transduction of primary rat intestinal cell cultures in vitro, or the rat colonic epithelium in vivo, with Ad-Pax-6 activated endogenous Proglucagon gene expression. These data demonstrate that Pax-6, but not Cdx-2/3, is capable of activating the endogenous Proglucagon gene in both immortalized enteroendocrine cells and the nontransformed intestinal epithelium in vivo.
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Pax-2 activates the Proglucagon gene promoter but is not essential for Proglucagon gene expression or development of Proglucagon-producing cell lineages in the murine pancreas or intestine.
Molecular endocrinology (Baltimore Md.), 2002Co-Authors: Grace Flock, Daniel J DruckerAbstract:Tissue-specific Proglucagon gene transcription is achieved through combinations of transcription factors expressed in pancreatic A cells and enteroendocrine L cells of the small and large intestine. Cell transfection and electrophoretic mobility shift assay experiments previously identified Pax-2 as a regulator of islet Proglucagon gene expression. We examined whether Pax-2 regulates gut Proglucagon gene expression using enteroendocrine cell lines and Pax2 1NEU mutant mice. Immunoreactive Pax-2 was detected in STC-1 enteroendocrine cells, and Pax-2 activated Proglucagon promoter activity in transfected baby hamster kidney and GLUTag cells. Pax-2 antisera diminished the formation of a Pax-2-G3 complex in electrophoretic mobility shift assay studies using nuclear extracts from islet and enteroendocrine cell lines. Surprisingly, Pax-2 mRNA transcripts were not detected by RT-PCR in RNA isolated from adult rat pancreas, rat islets, embryonic d 19 or adult murine pancreas and gastrointestinal tract. Furthermore, embryonic d 19 or neonatal d 1 Pax2 1NEU mice exhibited normal islet A cells and gut endocrine L cells, and no decrement in pancreatic or intestinal glucagon gene expression. These findings demonstrate that Pax-2 is not essential for the developmental formation of islet A or gut L cells and does not play a role in the physiological control of Proglucagon gene expression in vivo .( Molecular Endocrinology 16: 2349–2359, 2002)
Patricia L. Brubaker - One of the best experts on this subject based on the ideXlab platform.
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CLINICAL CASE SEMINAR Prolonged Gastrointestinal Transit in a Patient with a Glucagon-Like Peptide (GLP)-1- and-2-Producing Neuroendocrine Tumor
2016Co-Authors: Patricia L. Brubaker, Daniel J Drucker, Sylvia L Asa, Mark Redston, Carol Swallow, R. GreenbergAbstract:Neuroendocrine tumors overexpressing the Proglucagon-derived peptides have been associated with severe constipa-tion. The relationship between two of the intestinal proglu-cagon-derived peptides, glucagon-like peptide (GLP)-1 and-2, and delayed gastrointestinal transit, was characterized in a patient with a neuroendocrine Proglucagon-derived peptide tumor. A 60-yr-old female presented with intractable con-stipation and intermittent vomiting. Gastric, oral-ileal and colonic transit times, and plasma hormone levels were deter-mined before tumor resection. Expression of the Proglucagon-derived peptides by the tumor was determined by immuno-histochemistry, Northern blot analysis, HPLC, and RIA. Oral-cecal transit was more than 3 h, and a barium follow-through study showed dilated and thickened folds with most of the barium concentrated in the ileum at 24 h; residual bar-ium was identified in the colon at 14 d post ingestion. Circu-lating levels of GLP-1 and-2 were 300- to 400-fold elevated compared with levels in normal human subjects. Normal bowel function was restored by tumor resection. Consistent with the elevated plasma hormone levels, the tumor was found to express the Proglucagon gene, and immunoreactive GLP-1 and-2 were detected by both immunohistochemistry and RIA. Overexpression of glucagon-like peptide-1 and-2 is associated with markedly prolonged gastrointestinal transit in humans. These findings are consistent with a role for these peptides in the regulation of gastrointestinal motility. (J Clin Endocrinol Metab 87: 3078–3083, 2002) THE ILEAL BRAKE is a neurohormonal feedback mech-anism that delays gastric and intestinal transit time, thereby enhancing nutrient digestion and absorption in the proximal small intestine and preventing nutrient overflow into the distal gut (1). A number of gut peptides have been identified as possible effectors of intestinal motility (2), in
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Pax-6 Activates Endogenous Proglucagon Gene Expression in the Rodent Gastrointestinal Epithelium
Diabetes, 2003Co-Authors: Denny K.y. Trinh, Patricia L. Brubaker, Kai Zhang, Moazzem Hossain, Daniel J DruckerAbstract:The Proglucagon gene encodes pancreatic glucagon and the glucagon-like peptides, which exert diverse effects on nutrient absorption and assimilation. The therapeutic potential of glucagon-like peptide-1 (GLP-1) has fostered interest in development of cellular engineering approaches to augment endogenous intestinal-derived GLP-1 for the treatment of type 2 diabetes. We have used adenovirus technology to examine the potential roles of the transcription factors Cdx-2/3 and Pax-6 as activators of endogenous Proglucagon gene expression in enteroendocrine cell lines and in nontransformed rat intestinal cells. Adenoviral-expressed Cdx-2/3 and Pax-6 activated Proglucagon promoter-luciferase activity in baby hamster kidney (BHK) fibroblasts, HEK 293 cells, and enteroendocrine cell lines. Pax-6, but not Cdx-2/3, induced expression of the endogenous Proglucagon gene in enteroendocrine cell lines, but not in heterologous fibroblasts. Furthermore, transduction of primary rat intestinal cell cultures in vitro, or the rat colonic epithelium in vivo, with Ad-Pax-6 activated endogenous Proglucagon gene expression. These data demonstrate that Pax-6, but not Cdx-2/3, is capable of activating the endogenous Proglucagon gene in both immortalized enteroendocrine cells and the nontransformed intestinal epithelium in vivo.
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Transcriptional Activation of the Proglucagon Gene by Lithium and β-Catenin in Intestinal Endocrine L Cells
The Journal of biological chemistry, 2002Co-Authors: Younes Anini, Patricia L. Brubaker, Xianjun Fang, Gordon B. Mills, Tianru JinAbstract: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.
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Foxa3 (HNF-3gamma) binds to and activates the rat Proglucagon gene promoter but is not essential for Proglucagon gene expression.
Biochemical Journal, 2002Co-Authors: Yuanfang Liu, Wei Shen, Patricia L. Brubaker, Klaus H. Kaestner, Daniel J DruckerAbstract:Members of the Forkhead box a (Foxa) transcription factor family are expressed in the liver, pancreatic islets and intestine and both Foxa1 and Foxa2 regulate Proglucagon gene transcription. As Foxa proteins exhibit overlapping DNA-binding specificities, we examined the role of Foxa3 [hepatocyte nuclear factor (HNF)-3gamma] in control of Proglucagon gene expression. Foxa3 was detected by reverse transcriptase PCR in glucagon-producing cell lines and binds to the rat Proglucagon gene G2 promoter element in GLUTag enteroendocrine cells. Although Foxa3 increased rat Proglucagon promoter activity in BHK fibroblasts, augmentation of Foxa3 expression did not increase Proglucagon promoter activity in GLUTag cells. Furthermore, adenoviral Foxa3 expression did not affect endogenous Proglucagon gene expression in islet or intestinal endocrine cell lines. Although Foxa3(-/-) mice exhibit mild hypoglycaemia during a prolonged fast, the levels of Proglucagon-derived peptides and Proglucagon mRNA transcripts were comparable in tissues from wild-type and Foxa3(-/-) mice. These findings identify Foxa3 as a member of the Proglucagon gene G2 element binding-protein family that, unlike Foxa1, is not essential for control of islet or intestinal Proglucagon gene expression in vivo.
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prolonged gastrointestinal transit in a patient with a glucagon like peptide glp 1 and 2 producing neuroendocrine tumor
The Journal of Clinical Endocrinology and Metabolism, 2002Co-Authors: Patricia L. Brubaker, Daniel J Drucker, Sylvia L Asa, Carol J Swallow, Mark Redston, Gordon R GreenbergAbstract:Neuroendocrine tumors overexpressing the Proglucagon- derived peptides have been associated with severe constipation. The relationship between two of the intestinal Proglucagon-derived peptides, glucagon-like peptide (GLP)-1 and -2, and delayed gastrointestinal transit, was characterized in a patient with a neuroendocrine Proglucagon-derived peptide tumor. A 60-yr-old female presented with intractable constipation and intermittent vomiting. Gastric, oral-ileal and colonic transit times, and plasma hormone levels were determined before tumor resection. Expression of the Proglucagon-derived peptides by the tumor was determined by immunohistochemistry, Northern blot analysis, HPLC, and RIA. Oral-cecal transit was more than 3 h, and a barium follow-through study showed dilated and thickened folds with most of the barium concentrated in the ileum at 24 h; residual barium was identified in the colon at 14 d post ingestion. Circulating levels of GLP-1 and -2 were 300- to 400-fold elevated compared with levels in normal human subjects. Normal bowel function was restored by tumor resection. Consistent with the elevated plasma hormone levels, the tumor was found to express the Proglucagon gene, and immunoreactive GLP-1 and -2 were detected by both immunohistochemistry and RIA. Overexpression of glucagon-like peptide-1 and -2 is associated with markedly prolonged gastrointestinal transit in humans. These findings are consistent with a role for these peptides in the regulation of gastrointestinal motility.
Donald F. Steiner - One of the best experts on this subject based on the ideXlab platform.
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Impaired intestinal Proglucagon processing in mice lacking prohormone convertase 1.
Endocrinology, 2003Co-Authors: Randi Ugleholdt, Donald F. Steiner, Cathrine Orskov, Xiaorong Zhu, Carolyn F. Deacon, Jens J. HolstAbstract: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.
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Regulation of Proglucagon Transcription by Activated Transcription Factor (ATF) 3 and a Novel Isoform, ATF3b, through the cAMP-response Element/ATF Site of the Proglucagon Gene Promoter *
The Journal of biological chemistry, 2003Co-Authors: Jie Wang, Yun Cao, Donald F. SteinerAbstract:Glucagon, the second major glucose-regulated hormone in the control of glucose homeostasis, functions as a counter-regulator to insulin and is specifically produced by the pancreatic α cells. Its excessive biosynthesis and secretion is associated with diabetes mellitus. The expression of the Proglucagon gene has been demonstrated to be regulated by a cAMP-dependent pathway through cAMP-response element-binding protein (CREB) and possibly other transcription factors bound to its cAMP-response element (CRE)/activated transcription factor (ATF) site. Elsewhere we have shown that ATF3, a member of the ATF/CREB subfamily of the basic leucine zipper domain proteins, is expressed predominantly in the α cells of the pancreatic islets. In our attempts to further dissect the role of ATF3 proteins in α cells, we have identified and characterized a novel alternatively spliced form, ATF3b, and have compared the specific binding ability of ATF3 and ATF3b on the CRE/ATF motif of the Proglucagon promoter. Our findings indicate the existence of a novel mechanism by which the transcription of the Proglucagon gene is regulated in response to cAMP signals, in addition to CREB and in relation to glucose fluctuations in pancreatic α cells.
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severe defect in Proglucagon processing in islet a cells of prohormone convertase 2 null mice
Journal of Biological Chemistry, 2001Co-Authors: Machi Furuta, Gene C Webb, Raymond Carroll, An Zhou, Mariella Ravazzola, Lelio Orci, Donald F. SteinerAbstract:Abstract Mice homozygous for a deletion in the gene encoding prohormone convertase 2 (PC2) are generally healthy but have mild hypoglycemia and flat glucose-tolerance curves. Their islets show marked alpha (A)-cell hyperplasia, suggesting a possible defect in glucagon processing (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646–6651). In this report we have examined the biosynthesis and processing of Proglucagon in isolated islets from these mice via pulse-chase labeling and find that Proglucagon undergoes essentially no processing in chase periods up to 8 h in duration. Only a small percent of cleavage at the sensitive interdomain site (residues 71 and 72) appears to occur. These observations thus conclusively demonstrate the essentiality of PC2 for the production of glucagon in the islet A-cells. Ultrastructural and immunocytochemical studies indicate the presence of large amounts of Proglucagon in atypical appearing secretory granules in the hyperplastic and hypertrophic A-cells, along with morphological evidence of high rates of Proglucagon secretion in PC2 null islets. These findings provide strong evidence that active glucagon is required to maintain normal blood glucose levels, counterbalancing the action of insulin at all times.
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differential processing of Proglucagon by the subtilisin like prohormone convertases pc2 and pc3 to generate either glucagon or glucagon like peptide
Journal of Biological Chemistry, 1995Co-Authors: Yves Rouillé, Sean Martin, Donald F. SteinerAbstract:Proglucagon is processed differently in the islet α cells and the intestinal endocrine L cells to release either glucagon or glucagon-like peptide 1-(7-37) (GLP1-(7-37)), peptide hormones with opposing actions in vivo. In previous studies with a transformed α cell line (αTC1-6) we demonstrated that the kexin/subtilisin-like prohormone convertase, PC2 (SPC2), is responsible for generating the typical α cell pattern of Proglucagon processing, giving rise to glucagon and leaving unprocessed the entire C-terminal half-molecule known as major Proglucagon fragment or MPGF (Rouille, Y., Westermark, G., Martin, S. K., Steiner. D. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3242-3246). Here we present evidence, using mouse pituitary AtT-20 cells infected with a vaccinia viral vector encoding Proglucagon, that PC3 (SPC3), the major neuroendocrine prohormone convertase in these cells, reproduces the intestinal L cell processing phenotype, in which MPGF is processed to release two glucagon-related peptides, GLP1 and GLP2, while the glucagon-containing N-terminal half-molecule (glicentin) is only partially processed to oxyntomodulin and small amounts of glucagon. Moreover, in AtT-20 cells stably transfected with PC2 (AtT-20/PC2 cells), glicentin was efficiently processed to glucagon, providing further support for the conclusion that PC2 is the enzyme responsible for the α cell processing phenotype. In other cell lines expressing both PC2 and PC3 (STC-1 and βTC-3), Proglucagon was also processed extensively to both glucagon and GLP1-(7-37), although STC-1 cells express lower levels of PC2 and processed the N-terminal domain to glucagon less efficiently. In contrast, GH4C1 and COS 7 cells, which express very little or no PC2 or PC3, failed to process Proglucagon, aside from a low level of interdomain cleavage which occurred only in the GH4C1 cells. In vitro PC3 did not cleave at the single Arg residue in GLP1 to generate GLP1-(7-37), its truncated biologically active form, indicating the likelihood that another convertase is required for this cleavage.
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Proglucagon is processed to glucagon by prohormone convertase PC2 in alpha TC1-6 cells.
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Yves Rouillé, G Westermark, Sean K. Martin, Donald F. SteinerAbstract:Abstract Proglucagon is processed differentially in the pancreatic alpha cells and the intestinal L cells to yield either glucagon or glucagon-like peptide 1, respectively, structurally related hormones with opposing metabolic actions. Here, we have studied the processing of Proglucagon in alpha TC1-6 cells, an islet-cell line transformed by simian virus 40 large tumor (T) antigen, a model of the pancreatic alpha cell. We found that these cells process Proglucagon at certain dibasic cleavage sites to release glucagon and only small amounts of glucagon-like peptide 1, as demonstrated by both continuous and pulse-chase labeling experiments. Both normal islet alpha cells and alpha TC1-6 cells were shown to express the prohormone convertase PC2 at high levels, but not the related protease PC3. Expression of PC2 antisense RNA in alpha TC1-6 cells inhibited both PC2 production and Proglucagon processing concomitantly. We conclude that PC2 is the key endoprotease responsible for Proglucagon processing in cells with the alpha-cell phenotype.
Savita Dhanvantari - One of the best experts on this subject based on the ideXlab platform.
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Two Dipolar α-Helices within Hormone-encoding Regions of Proglucagon Are Sorting Signals to the Regulated Secretory Pathway
The Journal of biological chemistry, 2014Co-Authors: Leonardo Guizzetti, Rebecca Mcgirr, Savita DhanvantariAbstract:Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of Proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these Proglucagon-derived peptides. Our previous work suggested that Proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major Proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229–240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major Proglucagon fragment were sorted with different efficiencies, thus providing evidence that Proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of Proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting “signature.”
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The sorting of Proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals
The Journal of endocrinology, 2013Co-Authors: Rebecca Mcgirr, Leonardo Guizzetti, Savita DhanvantariAbstract:Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of Proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of Proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in Proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, Proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced Proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of Proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting Proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.
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Coregulation of glucagon-like peptide-1 synthesis with Proglucagon and prohormone convertase 1 gene expression in enteroendocrine GLUTag cells
Endocrinology, 2001Co-Authors: Savita Dhanvantari, Angelo Izzo, Erik Jansen, Patricia L. BrubakerAbstract:The insulinotropic hormone glucagon-like peptide-1 (GLP-1) is synthesized in the intestinal L cell by prohormone convertase 1 (PC1)-mediated posttranslational processing of Proglucagon. Previous studies have demonstrated that Proglucagon gene transcription in the L cell is stimulated by the protein kinase A (PKA) pathway through a cAMP response element (CRE). Because the PC1 gene contains two functional CREs, the present studies were conducted to investigate whether the PC1 and Proglucagon genes are coregulated by PKA, and to elucidate the temporal relationship(s) of PC1 and Proglucagon gene expression with production of GLP-1, in the intestinal cell. The GLUTag enteroendocrine cell line, which is known to express the Proglucagon gene and to synthesize and secrete GLP-1, was used as a model. Proglucagon and PC1 messenger RNA transcript levels were both increased after 12 h (but not 24 h) of treatment of GLUTag cells with forskolin/isobutylmethylxanthine (IBMX), by 2.7 +/- 0.3- and 2.4 +/- 0.3-fold, respectively, compared with controls (P < 0.01-0.001). Activation of PKA resulted in a 2.1 +/- 0.1-fold increase in PC1 reporter construct expression (P < 0.001) at 12 h, which was dependent on the presence of the CRE, and a 13- to 24-fold increment in PC1 protein levels (P < 0.01) at 12 and 24 h. Similarly, forskolin/IBMX increased secretion of GLP-1, by 1.8 +/- 0.2- and 2.2 +/- 0.6-fold at 12 and 24 h, respectively (P < 0.05-0.01). Although the cell content of GLP-1 was diminished after 12 h of treatment (P < 0.001), GLP-1 levels increased back to control values after 24 h of forskolin/IBMX treatment (P < 0.01 vs. 12-h levels). Thus, PKA-induced secretion of GLP-1 from the L cell is followed by restoration of the cellular peptide levels through a PKA-mediated, CRE-dependent up-regulation of Proglucagon and PC1 gene expression.
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Proglucagon processing in islet and intestinal cell lines
Regulatory peptides, 1996Co-Authors: J.devin Tucker, Savita Dhanvantari, Patricia L. BrubakerAbstract:To investigate the factors involved in the post-translational processing of Proglucagon, we have examined the Proglucagon-derived peptides (PGDPs) expressed in normal mouse pancreas and intestine, as well as in both islet (InR1-G9, RIN 1056A) and intestinal (STC-1) cell lines. N-terminal Proglucagon processing was similar to that of normal mouse pancreas in InR1-G9 cells, but differed in RIN 1056A and STC-1 cells, which contained significant amounts of glucagon as well as the intestinal PGDPs, glicentin and oxyntomodulin. The C-terminal end of Proglucagon was processed to small amounts of glucagon-like peptide-1 in InR1-G9 and RIN 1056A cells, as in normal pancreas, while processing was more extensive in both STC-1 cells and normal intestine. Northern blot analysis of mRNA transcripts for the prohormone convertases, PC1 and PC2, in the 3 cell lines demonstrated correlations between PC2 and the presence of glucagon, as well as between PC1 and production of the intestinal PGDPs. These findings provide support for the suggestion that PC1 and PC2 play roles in the tissue-specific post-translational processing of Proglucagon.
Tianru Jin - One of the best experts on this subject based on the ideXlab platform.
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Pbx1 is a co-factor for Cdx-2 in regulating Proglucagon gene expression in pancreatic A cells.
Molecular and cellular endocrinology, 2006Co-Authors: Tao Liu, Donald R Branch, Tianru JinAbstract:A number of Hox and Hox-like homeodomain (HD) proteins have been previously shown to utilize members of the TALE HD protein family as co-factors in regulating gene expression. The caudal HD protein Cdx-2 is a transactivator for the Proglucagon gene, expressed in pancreatic A cells and intestinal endocrine L cells. We demonstrate here that co-transfection of the TALE homeobox gene Pbx1 enhanced the activation of Cdx-2 on the Proglucagon promoter in either the pancreatic A cell line InR1-G9 or BHK fibroblasts. The activation was observed for Proglucagon promoter constructs with or without the binding motifs for Pbx1. Furthermore, mutating the penta-peptide motif (binding motif for TALE HD proteins) on Cdx-2 substantially attenuated its activation on Proglucagon promoter, but not on the sucrase-isomaltase gene (SI) promoter, or its own (Cdx-2) promoter; suggesting that Cdx-2 utilizes Pbx1 as a co-factor for regulating the expression of selected target genes. Physical interaction between Cdx-2 and Pbx1 was demonstrated by co-immunoprecipitation as well as GST fusion protein pull-down. We suggest that this study reveals a novel function for Pbx1 in pancreatic islet physiology: regulating Proglucagon expression by serving as a co-factor for Cdx-2.
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Redundant and synergistic effect of Cdx-2 and Brn-4 on regulating Proglucagon gene expression.
Endocrinology, 2005Co-Authors: Peixiang Wang, Tao Liu, Tianru JinAbstract:Cdx-2 and Brn-4 are recognized as transcriptional activators for the Proglucagon gene. These two homeodomain (HD) proteins are able to interact with the correspondent motifs on the G1 enhancer element of Proglucagon promoter, separated by only 8 bp. We have examined Brn-4 expression in Proglucagon-producing cells, isolated hamster Brn-4 cDNA, and localized its activation domain. Ectopic expression of either Cdx-2 or Brn-4 in the pancreatic B cell line In111 provoked it to express Proglucagon mRNA, whereas ectopically expressing both of them further stimulated Proglucagon mRNA expression in this cell line. Furthermore, Brn-4 was found to synergize with Cdx-2 in activating Proglucagon promoter, and the Brn-4 activation domain was not required for this synergistic activation. When the binding site for either Cdx-2 or Brn-4 was mutated, the synergistic activation by these two HD proteins was significantly attenuated, but not abolished. We propose that both cooperative DNA binding and mutual recruitment between Cdx-2 and Brn-4 are involved in this synergistic activation and have detected physical interaction between Cdx-2 and Brn-4 by glutathione-S-transferase-fusion protein pull-down assay. Our observations suggest that Cdx-2 and Brn-4, two HD proteins that belong to two different families, exert a synergistic and redundant effect on Proglucagon gene expression.
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Transcriptional Activation of the Proglucagon Gene by Lithium and β-Catenin in Intestinal Endocrine L Cells
The Journal of biological chemistry, 2002Co-Authors: Younes Anini, Patricia L. Brubaker, Xianjun Fang, Gordon B. Mills, Tianru JinAbstract: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.
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The Caudal Homeobox Protein cdx-2/3 Activates Endogenous Proglucagon Gene Expression in InR1-G9 Islet Cells
Molecular endocrinology (Baltimore Md.), 1997Co-Authors: Tianru Jin, D. K. Y. Trinh, Feng Wang, Daniel J DruckerAbstract: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 ...
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the caudal homeobox protein cdx 2 3 activates endogenous Proglucagon gene expression in inr1 g9 islet cells
Molecular Endocrinology, 1997Co-Authors: Tianru Jin, D. K. Y. Trinh, Feng Wang, Daniel J DruckerAbstract: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.
