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1.
Mol Metab ; 8: 144-157, 2018 02.
Article in English | MEDLINE | ID: mdl-29307512

ABSTRACT

OBJECTIVE: To characterize the EndoC-ßH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. METHODS: EndoC-ßH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. RESULTS: Transplantation of EndoC-ßH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-ßH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. CONCLUSIONS: Overall, the EndoC-ßH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-ßH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.


Subject(s)
Cell Culture Techniques/methods , Hypoglycemic Agents/pharmacology , Insulin-Secreting Cells/drug effects , Animals , Cell Line , Cells, Cultured , Diabetes Mellitus, Experimental/therapy , Drug Evaluation, Preclinical/methods , Humans , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Mice , Mice, SCID
2.
Regul Pept ; 175(1-3): 21-9, 2012 Apr 10.
Article in English | MEDLINE | ID: mdl-22252224

ABSTRACT

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands.


Subject(s)
Cell Membrane/metabolism , Glucagon-Like Peptide 1/pharmacology , Lung/metabolism , Receptors, Glucagon/metabolism , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Glucagon-Like Peptide-1 Receptor , Humans , Immunoblotting , Lung/cytology , Lung/drug effects , Mice , Molecular Sequence Data , Rabbits , Rats , Sequence Homology, Amino Acid , Swine
3.
J Biol Chem ; 285(1): 723-30, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19861722

ABSTRACT

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.


Subject(s)
Extracellular Space/metabolism , Glucagon-Like Peptide 1/chemistry , Receptors, Glucagon/chemistry , Amino Acid Sequence , Cell Line , Crystallography, X-Ray , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Glucagon/metabolism , Solutions
4.
Biochem Pharmacol ; 73(8): 1237-42, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17328868

ABSTRACT

The main purpose of this study was to examine the effects of a selective histamine H(3) receptor antagonist, NNC 38-1202, on caloric intake in pigs and in rhesus monkeys. The compound was given intragastrically (5 or 15 mg/kg), to normal pigs (n=7) and subcutaneously (1 or 0.1mg/kg) to obese rhesus monkeys (n=9). The energy intake recorded following administration of vehicle to the same animals served as control for the effect of the compound. In addition, rhesus monkey and pig histamine H(3) receptors were cloned from hypothalamic tissues and expressed in mammalian cell lines. The in vitro antagonist potencies of NNC 38-1202 at the H(3) receptors were determined using a functional GTPgammaS binding assay. Porcine and human H(3) receptors were found to have 93.3% identity at the amino acid level and the close homology between the monkey and human H(3) receptors (98.4% identity) was confirmed. The antagonist potencies of NNC 38-1202 at the porcine, monkey and human histamine H(3) receptors were high as evidenced by K(i)-values being clearly below 20 nM, whereas the K(i)-value on the rat H(3) receptor was significantly higher (56+/-6.0 nM). NNC 38-1202, given to pigs in a dose of 15 mg/kg, produced a significant (p<0.05) reduction (55%) of calorie intake compared with vehicle alone, (132.6+/-10.0 kcal/kgday versus 59.7+/-10.2 kcal/kgday). In rhesus monkeys administration of 0.1 and 1mg/kg decreased (p<0.05) average calorie intakes by 40 and 75%, respectively. In conclusion, the present study demonstrates that antagonistic targeting of the histamine H(3) receptor decreases caloric intake in higher mammalian species.


Subject(s)
Energy Intake/drug effects , Histamine Antagonists/pharmacology , Receptors, Histamine H3/physiology , Animals , Energy Intake/physiology , Humans , Macaca mulatta , Rats , Sus scrofa
5.
Eur J Pharmacol ; 532(1-2): 18-23, 2006 Feb 17.
Article in English | MEDLINE | ID: mdl-16448646

ABSTRACT

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.


Subject(s)
Receptors, Glucagon/genetics , Receptors, Glucagon/metabolism , Animals , Binding, Competitive , Blotting, Western , Caco-2 Cells , Calcium/metabolism , Cell Line , Cell Line, Tumor , Colforsin/pharmacology , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression , Glucagon-Like Peptide 2 , Glucagon-Like Peptide-2 Receptor , Glucagon-Like Peptides/pharmacology , Humans , Intestinal Mucosa/metabolism , Intestines/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
6.
Eur J Pharmacol ; 453(1): 33-41, 2002 Oct 18.
Article in English | MEDLINE | ID: mdl-12393057

ABSTRACT

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor.


Subject(s)
Receptors, Histamine H3/biosynthesis , Receptors, Histamine H3/genetics , Recombination, Genetic/physiology , Animals , Cell Line , Cloning, Molecular/methods , Histamine Agonists/metabolism , Histamine Antagonists/metabolism , Humans , Rats , Receptors, Histamine/metabolism
7.
Regul Pept ; 103(1): 9-15, 2002 Jan 15.
Article in English | MEDLINE | ID: mdl-11738243

ABSTRACT

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.


Subject(s)
Intestine, Large/growth & development , Intestine, Small/growth & development , Peptide Fragments/pharmacology , Receptors, Glucagon/agonists , Animals , Body Weight , Cell Line , Cricetinae , Cyclic AMP/metabolism , Drug Administration Schedule , Female , Glucagon-Like Peptide 2 , Glucagon-Like Peptide-1 Receptor , Glucagon-Like Peptides , Humans , Image Processing, Computer-Assisted , Injections, Subcutaneous , Intestine, Large/cytology , Intestine, Large/drug effects , Intestine, Small/cytology , Intestine, Small/drug effects , Mice , Mice, Inbred C57BL , Organ Size/drug effects , Peptide Fragments/chemistry , Protein Binding , Random Allocation , Receptors, Glucagon/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism , Transfection
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