Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the pancreas, ligands of receptor tyrosine kinases (RTKs) are thought to be implicated in the development and function of the islets of Langerhans, which represent the endocrine part of the pancreas. In a previous study, we randomly screened by reverse transcriptase-polymerase chain reaction for RTKs expressed in the embryonic pancreas. One cDNA fragment that was cloned during this screen corresponded to the KIT receptor. The objective of the present study was to analyze the pattern of Kit expression in the pancreas. We demonstrated that Kit is expressed and functional in terms of signal transduction in the insulin-producing cell line INS-1. Indeed, upon treatment with the KIT ligand (KITL), the extracellular signal-regulated protein kinase was phosphorylated, and the expression of early responsive genes was induced. We also demonstrated that Kit mRNAs are present in fetal and adult rat islets. We next used mice that had integrated the lacZ reporter gene into the Kit locus. In these mice, beta-galactosidase (beta-gal) served as a convenient marker for expression of the endogenous Kit gene. Kit was found to be specifically transcribed in beta-cells (insulin-expressing cells), whereas no expression was found in other endocrine cell types or in the exocrine tissue. Interestingly, not all mature beta-cells expressed Kit, indicating that Kit is a marker of a subpopulation of beta-cells. Finally, by following beta-gal expression in the pancreas during fetal life, we found that at E14.5, Kit is expressed in both insulin- and glucagon-expressing cells present at that stage, and also in a specific cell population present in the epithelium that stained negative for endocrine markers. These data suggest that these Kit-positive/endocrine-negative cells could represent a subpopulation of endocrine cell precursors.
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PMID:Expression of the receptor tyrosine kinase KIT in mature beta-cells and in the pancreas in development. 1152 67

In mammals, the COUP-TF-family consisting of two structurally related proteins, COUP-TFI and COUP-TFII belongs to the orphan member of the steroid/thyroid hormone receptor superfamily. In an attempt to gain insights into the role of COUP-TFII, we examined developmental expression pattern of the mouse COUP-TFII focusing our studies on endoderm-derived tissues, pancreas and liver in particular. Independent lines of transgenic mice expressing Escherichia coli beta-galactosidase driven by the COUP-TFII promoter were generated. Embryonic expression of the beta-gal protein at day 9 of gestation was detected in the notochord, the ventral neural tube and, interestingly, in the gut endoderm, a site where COUP-TFII has not been detected previously. Between 9.5 and 11.5 dpc, beta-gal expression pattern that was established earlier persisted and sections revealed a staining of the common atrial chamber of the heart. At 15.5 dpc, beta-gal activity was found in all endoderm-derived tissues. We found that COUP-TFII mRNA and protein were present in fetal and adult hepatocytes. Finally, COUP-TFII expression was detected in pancreas, as judged by co-expression of the beta-gal in some of the glucagon and PDX1 positive-cells at 12.5 dpc and co-expression with insulin positive-cells at 15.5 dpc. In adult pancreas, COUP-TFII protein was present in the endocrine islet cells.
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PMID:Expression of COUP-TFII in metabolic tissues during development. 1238 58

Embryonic stem (ES) cells can differentiate into many cell types and are expected to be useful for tissue engineering. Recent reports have shown that ES cells can differentiate into insulin-producing cells in response to the transient expression of the pdx-1 gene, after the removal of feeder cells. To investigate the lineage of insulin-producing cells and their in vitro differentiation, we introduced the betageo gene, encoding a beta-galactosidase-neomycin phosphotransferase fusion protein under the control of the mouse insulin 2 promoter, into ES cells that had been adapted to feeder-free culture, and analyzed insulin gene expression during their in vitro differentiation. We also examined the expression of transcription factors that are related to the differentiation of the pancreas. X-gal staining analysis revealed beta-galactosidase-positive cells on the surface and in the center of the embryoid body that proliferated during differentiation. Glucose-responsive insulin-producing cells, derived from our feeder-free ES cells, expressed insulin 2, pdx-1, Pax4, and Isl1 and also the glucagon, somatostatin, and PP genes. Moreover, the genes encoding p48, amylase, and carboxypeptidase A were also expressed. These results suggest that ES cells can differentiate not only into endocrine cells but also into exocrine cells of the pancreas, without the initiation of pdx-1 expression.
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PMID:Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells. 1271 47

In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular [Ca(2)+](i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor. In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells. The assay is very robust, with a Z' value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.
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PMID:A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists. 1459 49

Type 1 diabetes results from insulin deficiency caused by destruction of pancreatic beta cells. Glucagon-like peptide (GLP)-1 stimulates beta cell growth and differentiation. To determine whether continuous expression of GLP-1 in vivo can regenerate beta cells and remit type 1 diabetes in mice for a prolonged time, we constructed an adenoviral vector containing the cytomegalovirus promoter/enhancer and albumin leader sequence followed by GLP-1 cDNA (rAd-GLP-1). A single administration of rAd-GLP-1 via the tail vein into streptozotocin (STZ)-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in remission of diabetes within 10 days; normoglycemia remained until the experiment was terminated. The number of insulin-positive cells in the pancreas and insulin secretion significantly increased in rAd-GLP-1-treated mice compared with STZ-induced diabetic mice treated with rAd-beta-galactosidase. Glucose tolerance tests in mice that achieved normoglycemia after treatment with rAd-GLP-1 showed that the kinetics of glucose clearance was similar to normal NOD/SCID mice. Treatment of autoimmune diabetic mice with rAd-GLP-1 restored normoglycemia, which was maintained for 1 year when mice were also treated with an immunoregulator to halt the autoimmune response to beta cells. We suggest that regeneration of insulin-producing cells by GLP-1 gene therapy may be a potential method for prolonged control of type 1 diabetes in humans.
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PMID:Prolonged remission of diabetes by regeneration of beta cells in diabetic mice treated with recombinant adenoviral vector expressing glucagon-like peptide-1. 1716 79


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