UNIPROT:P01275 - FACTA Search

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Query: UNIPROT:P01275 (glucagon)
26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cryopreservation is the only available technique for long-term storage of pancreatic islets. The freezing/thawing protocol may cause considerable loss of viable islet tissue and impair its function in vivo. The aim of this study was to investigate glucose and insulin levels after transplantation of fresh and cryo/thawed rat islets. Rat pancreatic islets were isolated following intraductal collagenase injection and Ficoll gradient purification. After isolation, islets were cultured for 24 h and then either transplanted or frozen after stepwise addition of DMSO according to Rajotte et al. and stored in liquid nitrogen. After rapid thawing islets were stepwise transferred into RPMI medium and cultured for another 24 h. The recipients were athymic mice with streptozotocine-induced diabetes. Two hundred fresh (n=13) or cryo/thawed (n=15) islets were transplanted beneath the renal capsule. Glucose levels were measured for 14 days and blood samples for insulin determination were obtained 15 min after i.p. glucagon (10 mg/kg) administration on day 14. Glucose levels were normalized (<9 mmol/l) in all recipients within 3 days since transplantation. On day 14, mean fasting values+/-SE in fresh and cryo/thawed islet groups were 4.0+/-0.6 and 4.4+/-0.4 mmol/l, respectively (P>0.05). Fasting insulin levels were higher in the cryo/thaw than in the fresh islet group (1.67+/-0.33 vs 0.57+/-0.13 ng/ml; P<0.01). Post-glucagon levels did not differ significantly (1.45+/-0.24 vs 0.86+/-0.24 ng/ml; P=0.06). While glucagon significantly increased insulin levels (P<0.01) in the fresh islet group, no change in insulin levels was observed (P>0.05) in the cryo/thaw group. Immunohistochemical staining demonstrated fragmentation of viable islet tissue which was more apparent in the cryo/thaw group. We conclude that in a short-term study cryo/thawed rat islets produce higher insulin levels than fresh islets transplanted into nude mice. This may be due to better islet survival or loss of feed-back regulation.
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PMID:Increased glucagon-stimulated insulin secretion of cryopreserved rat islets transplanted into nude mice. 993 Sep 40

We assessed the potential role of all-trans-retinoic acid on the developing chick pancreas, specifically with regard to the proportions of insulin cells. The endodermal component of the dorsal pancreatic bud of 5-d-old chick embryos was cultured on Matrigel. Retinoic acid (10(-6) or 10(-5) M) was added to a standard serum-free medium, Ham's F12 containing insulin, transferrin and selenium (F12.ITS). Control grafts were cultured in F12.ITS alone or in F12.ITS with DMSO (the diluent for retinoic acid). After 7 d the explants were retrieved, freeze-dried, vapor-fixed, and embedded in resin. Endocrine cell types were identified by immunocytochemistry. The numbers of insulin cells were expressed as a proportion of the sum of insulin plus glucagon cells. Retinoic acid had a dose-related effect; the proportion of insulin cells in explants treated with the lower dose of retinoic acid (10(-6) M) was more than twice the proportion of insulin cells in explants treated with the higher dose (10(-5) M) of retinoic acid and more than three times that of the control grafts.
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PMID:The effect of retinoic acid on the proportion of insulin cells in the developing chick pancreas. 1069 Oct 36

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-junction proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser microscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most subapical plasma membrane at cell borders, while on the basolateral membrane both Cx32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most subapical plasma membrane and were colocalized with only Cx32-immunoreactive lines. In freeze-fracture analysis, many small gap-junction plaques were observed within a well-developed tight-junction strand network. The fence function of tight junctions in the cells, as examined by diffusion of labeled sphingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or anti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These results suggest that Cx32 gap junctions, but not those with Cx26, are closely coordinated with the expression and function of tight junctions in hepatocytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression.
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PMID:Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes. 1116 18

We are interested in the regulation of early pancreatic differentiation, particularly with regard to factors that enhance insulin cell proliferation. Both retinoic acid and insulin-like growth factor 1 (IGF-1) are known to be important in the proliferation and differentiation of insulin cells. Individually, they have the ability to increase the proportion of insulin cells when added to cultures of chick dorsal pancreatic buds. The aim of this study was to define the action of retinoic acid (RA) in combination with IGF-1 on the proportion of insulin cells. The dorsal pancreatic bud of 5-day-old chick embryos was excised and the endodermal component, with minimal adherent mesenchyme, was explanted onto Matrigel. RA (10(-6) M) and IGF-1 (50 ng/ml) were added to Ham's F12 culture medium containing transferrin (5 microg/ml) and selenium (10(-10) M) (F12.TS). Control explants were cultured in F12.TS alone or in F12.TS containing dimethyl sulphoxide (DMSO) [F12.TS (DMSO)]. After 7 days in culture, insulin and glucagon cells were localized immunocytochemically; numbers of insulin cells were expressed as a percentage of insulin plus glucagon cell counts. Addition of RA plus IGF-1 to the medium increased the proportion of insulin cells markedly (23.43%) compared with the proportions in control explants (11.3% with F12.TS (DMSO), 13.2% with F12.TS). This increase represents a more than twofold increase in the proportion of insulin cells over that of control explants.
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PMID:Regulation of embryonic chick insulin cells: effect of retinoic acid and insulin-like growth factor 1. 1134 Feb 60

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-7) M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.
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PMID:Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes. 1155 21

Glucagon-like peptide 1 (GLP-1) is an incretin hormone that is secreted from the enteroendocrine L-cells of the gut in response to nutrient ingestion. GLP-1 enhances both insulin secretion and insulin gene expression in a glucose-dependent manner via activation of its putative G-protein-coupled receptor on pancreatic beta-cells. In the presence of DMSO (0.5-2.5%), these functional responses were enhanced significantly (2- to 2.5-fold) in a concentration-dependent manner in the beta-cell line INS-1, although basal levels were not affected. Rat insulin 1 (rINS1) promoter activity appeared to be augmented in a cAMP-response element (CRE)-dependent manner as the effect of DMSO was abolished following a mutation in the CRE of the rINS1 promoter. Also, expression of a generic cAMP-driven reporter gene was enhanced by 1.5% DMSO in response to GLP-1 (3.5-fold), forskolin (2-fold), and 3-isobutyl-1-methylxanthine (2-fold). Analysis of intracellular signaling components revealed that DMSO did not elevate cAMP levels, protein kinase A activity, or phosphorylated levels of CRE-binding protein (CREB), CRE-modulator (CREM), and activating transcription factor-1 (ATF-1). These data suggest that GLP-1 induces insulin gene transcription in a CREB, CREM, and ATF-1-independent manner in beta-cells. The mechanism by which DMSO imparts this amplifying action is unclear but may involve redistribution of intracellular compartments or a direct molecular interaction with a downstream target of the GLP-1 receptor signaling pathway in the beta-cell. These effects of DMSO on incretin action may provide novel applications with respect to further characterizing GLP-1 receptor signaling, identifying incretin-like compounds in screening assays, and as a therapeutic treatment in type 2 diabetes.
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PMID:Synergistic effect of dimethyl sulfoxide on glucagon-like peptide 1 (GLP-1)-stimulated insulin secretion and gene transcription in INS-1 cells: characterization and implications. 1216 88

Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes (e.g., 102% with 1 micromol/L glucagon) and a simultaneous decrease in intracellular membranes (e.g., 38% with 1 micromol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion.
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PMID:Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytes. 1277 23

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

Both retinoic acid (RA) and transforming growth factor (TGF)-beta1 are known to be influential in the development of insulin cells. Respectively, they increase and decrease the proportion of insulin cells when added to cultures of embryonic chick dorsal pancreatic buds. The aim of this study was to define the action of RA in the presence of decreased levels of TGF-beta1, as are found in growth factor-reduced Matrigel (GFRM), on the proportion of insulin cells. The endodermal component of 5-d chick dorsal pancreatic buds was explanted on to GFRM. Retinoic acid (10(-6) M) was added to Ham's F12 culture medium containing insulin (5 microg/ml), transferrin (5 microg/ml), and selenium (10(-10) M) (F12.ITS). Control explants were cultured in F12.ITS alone or in F12.ITS containing dimethyl sulfoxide (DMSO). After 7 d in culture, insulin and glucagon cells were localized immunocytochemically; changes in numbers of insulin cells were expressed as a percentage of insulin plus glucagon cells. Medium containing RA or DMSO increased the proportion of insulin cells significantly compared with the proportion in the explants cultured in F12.ITS medium alone.
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PMID:Regulation of the proportion of insulin cells in embryonic chick pancreas: effect of a growth factor-reduced extracellular matrix in combination with retinoic acid. 1461 34

A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (54-68 kg) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10(-6) or 10(-7) M), linoleic acid (3.4 x 10(-5) M), and carnitine (10(-3) M) for 3 days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1, or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 h; medium and all cells were harvested on the third day. Concentrations of acetoacetate and beta-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate:beta-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 h, over the range of insulin, glucagon, and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine.
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PMID:The role of insulin, glucagon, dexamethasone, and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs. 1521 32

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