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)

Fetal calf serum and a beta-cell line exhibit a proteolytic activity essential for the biological function of glucagon-like peptide-1 (GLP-1). This process of cleavage was investigated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To generate processing products, GLP-1 was subjected to rat insulinoma m5F (RINm5F) cell cultures or to fetal calf serum (FCS). For detection of processing products, a standardized extraction method including ion-exchange batch extraction, ultrafiltration, gel filtration, and reversed-phase chromatography was used. The RP fractions were analyzed by MALDI-TOF-MS. Processed proteolytic products were detected by comparing the resulting mass spectra of cell media or FCS after 2 h incubation with GLP-1 (7-36) amide with these of 2 h controls. To perform the comparison of the resulting mass spectra, software (MASSSPECANALYST) based on Microcal Software, Origins C-like language LABTALK was developed. GLP-1 fragments were purified by RP-HPLC, and characterized by sequence analysis. As insulin is the major secretory product of beta cells depending on GLP-1 stimulation, the insulin and insulin fragments of the cell culture supernatants were also analyzed by this method.
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PMID:Proteolytic cleavage of glucagon-like peptide-1 by pancreatic beta cells and by fetal calf serum analyzed by mass spectrometry. 1048 Feb 53

Glucagon-like peptide-1-(7-36) (GLP-1) is a hormone derived from the proglucagon molecule, which is considered a highly desirable antidiabetic agent mainly due to its unique glucose-dependent stimulation of insulin secretion profiles. However, the development of a GLP-1-based pharmaceutical agent has a severe limitation due to its very short half-life in plasma, being primarily degraded by dipeptidyl peptidase IV (DPP-IV) enzyme. To overcome this limitation, in this article we propose a novel and potent DPP-IV-resistant form of a poly(ethylene glycol)-conjugated GLP-1 preparation and its pharmacokinetic evaluation in rats. Two series of mono-PEGylated GLP-1, (i) N-terminally modified PEG(2k)-N(ter)-GLP-1 and (ii) isomers of Lys(26), Lys(34) modified PEG(2k)-Lys-GLP-1, were prepared by using mPEG-aldehyde and mPEG-succinimidyl propionate, respectively. To determine the optimized condition for PEGylation, the reactions were monitored at different pH buffer and time intervals by RP-HPLC and MALDI-TOF-MS. The in vitro insulinotropic effect of PEG(2k)-Lys-GLP-1 showed comparable biological activity with native GLP-1 (P = 0.11) in stimulating insulin secretion in isolated rat pancreatic islet and was significantly more potent than the PEG(2k)-N(ter)-GLP-1 (P < 0.05) that showed a marked reduced potency. Furthermore, PEG(2k)-Lys-GLP-1 was clearly resistant to purified DPP-IV in buffer with 50-fold increased half-life compared to unmodified GLP-1. When PEG(2k)-Lys-GLP-1 was administered intravenously and subcutaneously into rats, PEGylation improved the half-life, which resulted in substantial improvement of the mean plasma residence time as a 16-fold increase for iv and a 3.2-fold increase for sc. These preliminary results suggest a site specifically mono-PEGylated GLP-1 greatly improved the pharmacological profiles; thus, we anticipated that it could serve as potential candidate as an antidiabetic agent for the treatment of non-insulin-dependent diabetes patients.
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PMID:Synthesis, characterization, and pharmacokinetic studies of PEGylated glucagon-like peptide-1. 1576 92

Inhibition of dipeptidyl peptidase IV (DPP-IV) activity has been reported to improve nutrient-stimulated insulin secretion through the stabilization of glucagon-like peptide (GLP-1). In the present study, we identified novel DPP-IV inhibitors of pyrazolidine derivatives (Compounds 1 and 2) and characterized their biological effects in vitro and in vivo. Compound 1, an isoleucine pyrazolidide with a phenyl urea group, inhibited rat plasma DPP-IV, porcine kidney DPP-IV, as well as human Caco-2 DPP-IV with IC(50) values of 1.70, 2.26, and 2.02 microM, respectively. Because of the poor pharmacokinetic properties of Compound 1, further optimization was carried out, leading to the discovery of Compound 2, which had similar in vitro activities. Compound 2 acted as a selective and competitive inhibitor of DPP-IV. MALDI-TOF mass spectrometric analysis proved that the compound (20 microM) effectively blocked the degradation of active GLP-1 peptide by 61%. Although similar in in vitro potency, marked improvement of in vivo efficacy and pharmacokinetic properties was seen with Compound 2. Oral administration of Compound 2 resulted in potent and rapid inhibition of circulating DPP-IV in C57BL/6J mice, with ED(50) values of 26mg/kg (s.c.) and 42mg/kg (p.o.). In addition, this compound improved glucose tolerance in ob/ob mice, as determined by an oral glucose tolerance test (OGTT). These results indicate that Compound 2 is a potent and selective DPP-IV inhibitor with oral anti-hyperglycemic activity in vivo.
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PMID:Inhibition of dipeptidyl peptidase IV by novel inhibitors with pyrazolidine scaffold. 1589 94

Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat alpha-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.
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PMID:Expression, purification, and characterization of C-terminal amidated glucagon in Streptomyces lividans. 1860 50

This paper describes the hyphenation of cIEF and MALDI-TOF MS via a fractionation or spotting device. After focusing in cIEF the compounds are hydrodynamically mobilized and deposited on a MALDI target plate using a sheath liquid interface, which provides the catholyte solution and the electrical ground. From previous experiments, sample conditions that resulted in a high resolution in cIEF and acceptable protein signal intensity in MS were selected [Silvertand et al., Electrophoresis, 2008, 29, 1985-1996]. Besides the mixture of test proteins, the sample solution contains 1% Pharmalyte, 0.3% hydroxyethyl cellulose and 0.1% Tween 20 and is used for both optimization as well as characterization of the cIEF-MALDI-TOF MS system. Hyphenation problems encountered are mainly due to transfer of the liquid from the needle to the MALDI target plate and are solved by choosing the proper sheath catholyte (200 mM NH4OH in 50% methanol with 0.1% Tween20). MS electropherograms were reconstructed by plotting the intensities of the m/z values corresponding to the proteins versus migration time (related to spot number). Reproducibility, peak width and signal intensity for different focusing and spotting (fractionation) times were calculated using these reconstructed MS electropherograms as well as the UV electropherograms. The best results were obtained with focusing time of 75 min (no under- or overfocusing) and a spotting time of 5 s (highest protein signal intensity in MS). The applicability of the system is demonstrated by the analysis of a biopharmaceutical (glucagon) and its deamidation product.
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PMID:Development and characterization of cIEF-MALDI-TOF MS for protein analysis. 1939 Nov 48

To date there have been few published immunoassays for the important iron regulator hepcidin. This study describes a novel competitive radioimmunoassay (RIA) for the bioactive hepcidin peptide. A rabbit anti-hepcidin polyclonal antibody was produced using synthetic hepcidin radiolabelled with 125I to produce a competitive RIA. Normal patient (n=47) samples were collected and assayed for hepcidin to determine a reference range. Other patient groups collected were ulcerative colitis (UC; n=40), iron deficiency anaemia (IDA; n=15), chronic kidney disease not requiring dialysis (CKD; n=45) and chronic kidney disease requiring dialysis (HCKD; n=94). Detection limit of the assay was determined as 0.6 ng/mL. Intra-assay precision was 5 ng/mL (7.2%) and 50 ng/mL (5.8%), interassay precision was 5 ng/mL (7.6%) and 50 ng/mL (6.7%). Analytical recovery was 98% (5 ng/mL), 94% (10 ng/mL) and 97% (50 ng/mL). The assay was linear up to 200 ng/mL. No demonstrable cross-reactivity with human insulin, glucagon I, angiotensinogen I, beta-defensin 1-4, alpha-defensin-1 and plectasin was observed. There was significant correlation (r=0.96, P < or = 0.0001) between the hepcidin RIA and an established hepcidin SELDI-TOF-MS method. Analysis of the normal human samples gave a reference range of 1.1-55 ng/mL for hepcidin. Further statistical evaluation revealed a significant difference between male and female hepcidin levels. There was significant correlation between hepcidin and ferritin in the control group (r=0.6, P < or = 0.0001). There was also a significant difference between the normal and disease groups (P < or = 0.0001). Healthy volunteers (n=10) showed a diurnal increase in plasma hepcidin at 4.00 pm compared to 8.00 am. A robust and optimised immunoassay for bioactive hepcidin has been produced and the patient sample results obtained further validates the important role of hepcidin in iron regulation, and will allow further investigation of this important peptide and its role in iron homeostasis.
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PMID:Development of a novel immunoassay for the iron regulatory peptide hepcidin. 1983 27

Motion sickness is caused by exposure to unfamiliar motions and typical symptoms of motion sickness include nausea and vomiting. To observe the metabolic and hormonal differences between nausea/vomiting (NAV) subjects and non-nausea/vomiting (NNV) ones, and to understand how the differences in metabolites and hormones affect the tolerance of organism to acceleration, 60 volunteers were exposed to repetitive acceleration using a 6-degree-of-freedom ship motion simulator. Meanwhile, 36 rats were randomly divided into three groups: an acceleration model group (n=14, exposed to acceleration), insulin group (n=14, intraperitoneal injection of insulin 30 min before exposure to acceleration), and control group (n=8). Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF/MS) was applied to analyze the serum metabolites in human subjects. Serum glucocorticoid, insulin, and glucagon levels were determined by radioimmunoassay in the NAV and NNV subjects as well as in rats, and serum epinephrine level was determined by ELISA. After acceleration exposure, 9 metabolites, including L-histidine, L-ornithine, L-serine, L-tyrosine, pyroglutamic acid, fumaric acid, urea, n-dodecanoic acid and n-tetradecanoic acid, had different changes in the NAV and NNV groups. The serum levels of 4-hydroxy-L-proline, glucose, oleic acid and urea were significantly higher in the NAV group than in the NNV group after exposure; however, only the elevation degree of serum glucose was significantly greater in the NAV group than in the NNV group (P<0.05). Serum cortisol and epinephrine were increased in both groups. Before exposure, insulin level in the NAV group was significantly lower than that in the NNV group (P<0.05). After rotation exposure, rat serum glucose in the insulin group was significantly lower than that in the acceleration model group (P<0.001), and the motion sickness index was significantly lower than that in the acceleration model group (P<0.05). Our study provides the first evidence that stable glucose level can help to relieve gastrointestinal symptoms in motion sickness, and suggests that acute hyperglycemia is related to gastrointestinal symptoms in motion sickness.
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PMID:Acute hyperglycemia is related to gastrointestinal symptoms in motion sickness: an experimental study. 2190 24

Glucagon-like peptide-1 (GLP-1)(2) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40 g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.
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PMID:High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production. 2277 32

Boc5, the first nonpeptidic agonist of Glucagon-like peptide-1 receptor, has been recognized as a potential candidate for treatment of diabetes. However, the metabolic behaviors of this novel molecule in both human and experimental animals remain unclear. This study aimed to explore the metabolic behaviors of Boc5 in biological preparations from human, pig and rat. Boc5 was found to be very stable in liver microsomes of human, pig and rat, but it can be degraded to two metabolites in plasma from all three species, via the successive hydrolysis of the C-22 esters. Chemical inhibition studies using selective esterase inhibitors and assays with purified enzymes suggested that Boc5 hydrolysis in human was totally mediated by human serum albumin (HSA) rather than esterases. ESI-TOF-MS/MS analysis revealed that Lys525 of HSA could be modified by treatment with Boc5, strongly suggesting the pseudo-esterase activity of albumin. Studies on species differences in this albumin-mediated metabolism showed large species differences in degradation rate of Boc5, the half lives of Boc5 in plasma from three various species varied from 23.5 h to 83.1h, but they were much closer to the half lives of Boc5 in corresponding serum albumins, implying the predominant role of serum albumin in plasma metabolism of Boc5. Additionally, the effects of various ligands including fatty acids and several drugs with unambiguous binding sites on HSA, on the pseudo-esterase activity of HSA, were also investigated using both experimental and molecular modelling studies. These results showed that the binding of various ligands to HSA could significantly affect the pseudo-esterase activity of HSA towards Boc5, due to the ligand-induced conformation changes of HSA.
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PMID:The role of serum albumin in the metabolism of Boc5: molecular identification, species differences and contribution to plasma metabolism. 2322 Apr 86

The rapid degradation of porcine glucagon-like peptide-2 (pGLP-2) by the enzyme dipeptidyl peptidase-IV (DPP-IV) is the main impediment in the development of pGLP-2 as a potential therapeutic agent for intestinal dysfunction and damage. In this study, one mono-modified Lys(30)-polyethylene glycol (PEG)-pGLP-2 was prepared using mPEG-succinimidyl propionate. To determine the optimized condition for PEGylation, the reactions were monitored by RP-HPLC and MALDI-TOF-MS. Stability was tested in purified DPP-IV in vitro. In vivo, the protective effects for colonic injury were measured in dextran sulfate sodium (DSS)-induced colitis in mice. The monoPEGylated products reached the maximum yield at 4:1 ratio of mPEG5k-SPA to pGLP-2. An effective method of successfully separating PEGylated pGLP-2 from mPEG-SPA5kD using CM Sepharose Fast Flow resin was established. The half-life of Lys(30)-PEG-pGLP-2 was 16-fold longer than that of pGLP-2 in DPP-IV. The DSS mice exhibited marked weight loss), which was significantly reduced by Lys(30)-PEG-pGLP-2 therapy. DSS treatment significantly increased colonic damage score, which was significantly reduced by administration of Lys(30)-PEG-pGLP-2 in DSS-mice. DSS-induced colitis clearly induced Myeloperoxidase activity in the colon, which was significantly reduced by treatments with 3% DSS-pGLP-2 or 3% DSS-PEG-pGLP-2. These results showed that site-specific Lys(30)-PEG-GLP-2 was resistant to degradation and reduced the severity of colonic injury in murine colitis. The enhanced biological potency of this product highlighted its potential as a therapeutic agent for intestinal diseases.
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PMID:Purified PEGylated porcine glucagon-like peptide-2 reduces the severity of colonic injury in a murine model of experimental colitis. 2427 71


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