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H C Fehmann

Publications and source records attributed to H C Fehmann.

At least 73 records · Page 4Linked to original sources
PubMed1994

Interaction of glucagon-like peptide-I (7-37) and somatostatin-14 on signal transduction and proinsulin gene expression in beta TC-1 cells.

The interactions of glucagon-like peptide-I(7-37)/(7-36)amide (GLP-I) and somatostatin-14 were characterized on the cyclic adenosine monophosphate (cAMP)-dependent signal transduction pathway and on proinsulin gene expression using mouse insulinoma beta TC-1 cells. GLP-I stimulated the activity of adenylate cyclase maximally at 1 mumol/L (151%). This effect was inhibited by 1 mumol/L somatostatin (119%). Forskolin also stimulated adenylate cyclase activity (10 mumol/L forskolin, 265%), and this action was inhibited by somatostatin (220%). Somatostatin alone left the basal adenylate cyclase activity unaltered. Somatostatin reduced the GLP-I-stimulated increase of intracellular cAMP levels (100 nmol/L GLP-I, 141%; 100 nmol/L GLP-I + 1 mumol/L somatostatin, 110%). GLP-I stimulated concentration-dependently the activity of protein kinase A (PKA), with a maximum at 10 nmol/L (181%). This action was inhibited by 100 nmol/L somatostatin (118%), but somatostatin did not influence the basal PKA activity. Furthermore, somatostatin reduced the GLP-I-induced stimulation of proinsulin gene expression (10 nmol/L GLP-I, 176%; 10 nmol/L GLP-I + 1 mumol/L somatostatin, 77%). Somatostatin itself inhibited concentration-dependently proinsulin gene expression (1 mumol/L somatostatin, 53%). These data demonstrate that GLP-I increases the activities of both adenylate cyclase and cAMP-dependent PKA, whereas somatostatin counteracts the stimulatory effect of GLP-I on adenylate cyclase activity, cAMP generation, PKA activity, and proinsulin gene expression. The interaction of both hormones occurs at the level of adenylate cyclase. Therefore, the interaction of both peptide hormones regulates downstream events, including gene expression.

Adenylyl Cyclases↗
PubMed1994

Stable expression of the rat GLP-I receptor in CHO cells: activation and binding characteristics utilizing GLP-I(7-36)-amide, oxyntomodulin, exendin-4, and exendin(9-39).

Glucagon-like peptide-I (GLP-I) is a potent insulinotropic peptide that mediates its actions at pancreatic B-cells via specific receptors. In the present study we stably expressed the rat B-cell GLP-I receptor in CHO cells and studied binding characteristics and receptor activation utilizing the naturally occurring receptor agonist GLP-I(7-36)-amide (GLP-I), the proglucagon-derived GLP-I-related peptide oxyntomodulin, the GLP-I receptor agonist exendin-4, and the specific antagonist exendin(9-39). The potencies to displace [125I]GLP-I from the receptor were GLP-I > exendin-4 > exendin(9-39) > oxyntomodulin, and to displace [125I]exendin-4 GLP-I = exendin-4 > exendin(9-39) > oxyntomodulin. cAMP production was stimulated equally by GLP-I and exendin-4. Oxyntomodulin was less potent to stimulate cAMP generation. Exendin(9-39) blocked the stimulatory action of GLP-I and exendin-4 on cAMP production, but not that of oxyntomodulin. This study shows that GLP-I and exendin-4 are potent agonists at the transfected rat B-cell GLP-I receptor whereas oxyntomodulin is only a weak GLP-I receptor agonist. Furthermore, exendin(9-39) is a potent GLP-I receptor antagonist. This peptide is a valuable tool to further study the physiological actions of GLP-I.

Animals↗
PubMed1994

Molecular cloning of a cDNA encoding for the GLP-1 receptor expressed in rat lung.

Recent data revealed the existence, localization and possible function of specific receptors for glucagon-like peptide 1 (7-36) amide (GLP-1) in rat lung. This receptor has different biochemical features than the GLP-1 receptor in endocrine pancreas. Therefore, we aimed to clone the lung receptor cDNA in order to analyze whether biochemical and functional diversity of the GLP-1 receptors in lung and pancreas is based upon genetic differences. A cDNA library from rat lung in a lambda gt11 vector was screened with a cDNA probe coding for the rat pancreas GLP-1 receptor. Thereby, we found a lung GLP-1 receptor cDNA which shows nearly complete homology to the pancreatic beta-cell receptor cDNA. Only one base exchange occurred at base 1 of a codon at position 977 resulting in a change of valine residue for isoleucine at position 323 of the amino acid sequence within the fifth transmembrane region. Northern blot hybridization identified transcripts at 2.7, 3.4, and 3.6 Kb. Expression of the recombinant lung GLP-1 receptor cDNA in CHO cells displayed a pharmacological profile similar to that seen with cells expressing the beta-cell derived cDNA. Therefore, we conclude that tissue-specificity for GLP-1 receptors is based upon posttranslational modifications of the receptor protein (for example glycosilation) or alternative splicing of primary transcripts and not on variations within the coding sequence of the receptor gene.

Amino Acid Sequence↗
PubMed1994

Expression of the ras-related rab3a gene in human insulinomas and normal human pancreatic islets.

To determine the expression of the small-molecular-weight guanosine 5'-triphosphate (GTP)-binding protein rab3a in human endocrine pancreatic tissue, total RNA was isolated from five different human insulinomas and from normal human islets. The expression of rab3a was analyzed by Northern blots utilizing a 1,300-bp Eco RI fragment of the cDNA coding for human rab3a. As in the brain, a specific transcript of 1,800 bp was detected in all insulinomas, though with varying signal intensity. When the northern membranes were rehybridized with a radioactively labeled insulin probe, a signal was found for all five insulinomas and cells of normal islets. In contrast, normal human islets showed no detectable expression of rab3a. In conclusion, rab3a is expressed to a significant degree in human insulinoma tissue but not in normal islets.

Animals↗
PubMed1994

Intestinal effects of alpha-glucosidase inhibitors: absorption of nutrients and enterohormonal changes.

The present paper addresses the question how alpha-glucosidase inhibitors affect glucose homeostasis. To facilitate this already established data on the effects of induced malabsorption on gut hormones such as gastric inhibitory polypeptide (GIP) in connection with preliminary findings which deal with the new incretin hormone glucagon-like peptide 1 (7-36) amide (GLP-1) are discussed. To emphasize the possibly important impact of a regulated GLP-1 release in response to glucosidase inhibitor treatment we evaluate the recently introduced concept of 'glucose competence' of pancreatic beta-cells. The slowing of nutrient (i.e. glucose) absorption by therapeutic means (for example, acarbose) could supplement a new approach in the treatment of type 2 diabetics which would utilize the well-preserved insulinotropic activity of GLP-1 in these patients, its glucagon-lowering effect, and its possible inhibition of gastric emptying rates, the latter helping to reduce the requirement for rapid insulin secretory responses as is intended while using alpha-glucosidase inhibitor treatment.

Animals↗
PubMed1994

Detection of the human glucagon-like peptide 1(7-36) amide receptor on insulinoma-derived cell membranes.

125I-glucagon-like peptide 1(7-36)amide was covalently cross-linked to a specific binding protein in human insulinoma cell membranes. A single radiolabeled band at M(r) 63,000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex. The molecular weight of this apparent GLP-1 receptor in human endocrine pancreatic tissue was of identical size as the GLP-1 receptor on rat insulinoma-derived RINm5F cell membranes. The radiolabeled band was undetectable when 1 microM of unlabeled GLP-1(7-36)amide or of the GLP-1 antagonist exendin(9-39)amide was included in the binding assay. Utilizing isolated poly-A+ RNA from the human insulinoma and a 1,500 bp Eco-RI fragment of the cDNA coding for the rat GLP-1(7-36)amide receptor for Northern blot analysis, a main hybridization signal at about 7 kb was found by Northern blotting. Our data provide the first direct evidence of the existence of GLP-1 receptors in human endocrine pancreatic tissue.

Animals↗
PubMed1994

Molecular and functional characterization of insulin receptors present on hamster glucagonoma cells.

Studies using pancreas perfusion techniques point to a physiological inhibition of glucagon release by insulin which should be mediated by A cell-residing insulin receptors. In this study, we have characterized the insulin receptors expressed in a hamster glucagonoma A cell line (INR1G9 cells) which is an accepted tool for A cell studies. In receptor binding assays 125I-insulin was displaced with a Kd of 3 nmol/l. Binding was also dependent upon time, temperature and cell number. Insulin concentration-dependently inhibited glucagon secretion (1 mumol: 59%, 100 nmol/l: 71%, 10 nmol/l: 86% of controls). In transient transfection experiments insulin inhibited proglucagon gene transcription (controls: 100%, 100 nmol/l: 54%, 10 nmol/l: 57%, 1 nmol/l: 72%, 100 pmol/l: 96%). Treatment of INR1G9 cells with insulin for 20 h induced a strong downregulation of insulin receptors (controls: 100%, 100 nmol/l: 30%, 10 nmol/l: 70%, 1 nmol/l: 73%, 100 pmol/l: 75%) and of insulin receptor mRNA levels (controls: 100%, 100 nmol/l: 42%, 10 nmol/l: 82%, 1 nmol/l: 84%, 100 pmol/l: 90%). When INR1G9 cells were transiently transfected with a hybrid gene containing the promotor/enhancer region of the human insulin receptor promotor (1,462 bp) linked to the transcriptional reporter gene chloramphenicol acetyltransferase and were treated with insulin it was demonstrated that insulin did not affect the insulin receptor gene transcription. In conclusion, INR1G9 cells express specific receptors for insulin. Insulin inhibits glucagon secretion and proglucagon gene expression via an inhibition of proglucagon gene transcription. Ligand-induced downregulation of the insulin receptor is not mediated by changes of insulin receptor gene transcription and is most likely regulated by posttranscriptional mechanisms, e.g. destabilization of insulin receptor mRNA.

Animals↗
PubMed1994

Ligand-specificity of the rat GLP-I receptor recombinantly expressed in Chinese hamster ovary (CHO-) cells.

Glucagon-like peptide-I (GLP-I) is a potent incretin hormone and is considered as a new therapeutic tool in the treatment of diabetes mellitus. This study was designed to precisely characterize the binding behavior and activation of the recombinant GLP-I receptor against naturally occurring ligands of the glucagon/VIP/secretin peptide hormone family. CHO-cells were stably transfected with a plasmid containing a cDNA encoding for the rat GLP-I receptor. Northern blot analysis with this cDNA showed a single band of 2.7 kb in CHO cells, while in RINm5F cells, three bands of 2.7, 3.4, and 3.6 kb were specifically labelled. In receptor-binding studies 125I-GLP-I was displaced by GLP-I and weakly by PHI and oxyntomodulin but not by helodermin, helospectin I, helospectin II, secretin, VIP, and PACAP-38. Intracellular cAMP generation was stimulated by GLP-I, PHI, and oxyntomodulin. Helodermin, helospectin I, helospectin II, secretin, VIP, and PACAP-38 were not able to displace 125I-GLP-I from its receptor or to stimulate intracellular cAMP production. This data shows that the GLP-I receptor is characterized by a high ligand specificity.

Animals↗
PubMed1993

Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells.

Exendin-4 purified from Heloderma suspectum venom shows structural relationship to the important incretin hormone glucagon-like peptide 1-(7-36)-amide (GLP-1). We demonstrate that exendin-4 and truncated exendin-(9-39)-amide specifically interact with the GLP-1 receptor on insulinoma-derived cells and on lung membranes. Exendin-4 displaced 125I-GLP-1, and unlabeled GLP-1 displaced 125I-exendin-4 from the binding site at rat insulinoma-derived RINm5F cells. Exendin-4 had, like GLP-1, a pronounced effect on intracellular cAMP generation, which was reduced by exendin-(9-39)-amide. When combined, GLP-1 and exendin-4 showed additive action on cAMP. They each competed with the radio-labeled version of the other peptide in cross-linking experiments. The apparent molecular mass of the respective ligand-binding protein complex was 63,000 Da. Exendin-(9-39)-amide abolished the cross-linking of both peptides. Exendin-4, like GLP-1, stimulated dose dependently the glucose-induced insulin secretion in isolated rat islets, and, in mouse insulinoma beta TC-1 cells, both peptides stimulated the proinsulin gene expression at the level of transcription. Exendin-(9-39)-amide reduced these effects. In conclusion, exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP-1 receptor antagonist.

Animals↗
PubMed1993

Glucose-dependency of the insulin stimulatory effect of glucagon-like peptide-1 (7-36) amide on the rat pancreas.

The glucose-dependent action of GLP-1 (7-36) amide (GLP-1) on insulin secretion was studied in isolated islets and in the perfused rat pancreas. In islet experiments in the presence of non-stimulatory glucose levels (< 3 mmol/l) a GLP-1 concentration of 10 nmol/l increased insulin secretion by 83%. However, higher GLP-1 concentrations (25 and 100 nmol/l) could not further enhance this effect (85 and 83%, respectively). The onset of the stimulatory action of a supramaximal GLP-1-load (25 nmol/l) was at a glucose level of 3 mmol/l. In the perfused pancreas, 25 nmol/l GLP-1 induced a strong insulin release at 5 mmol/l glucose, but under basal glucose (2.8 mmol/l) only a slight enhancement of insulin secretion occurred during the late phase (30 to 54 min) of perfusion (P < 0.05). In conclusion, a slight but not dose-dependent stimulation of insulin secretion by supramaximal GLP-1 loads under basal glucose levels was found. The necessary GLP-1 concentrations to achieve this in vitro effect are beyond physiological or postprandial levels.

Amides↗
PubMed1993

The stimulatory effect of amylin on mucus secretion in isolated rat trachea.

A modified Ussing chamber technique was used to characterize the effects of amylin on rat tracheal mucus secretion. The effects of amylin were studied at both the mucosal and the submucosal side of isolated rat trachea. Applied at the mucosal side, amylin had no direct effect on mucus secretion, but significantly decreased the acetylcholine (1 mM)-induced tachyphylaxia when this substance was presented a second time (first stimulation: 490%; second stimulation: 334%; second stimulation plus amylin: 407%). At the submucosal side, amylin significantly stimulated mucus secretion (controls: 100%; 1 microM amylin: 193%; 100 nM amylin: 170%; 10 nM amylin: 127%; 1 nM amylin: 127%). Together with the recent observation of specific amylin receptors in rat lung membranes, these data suggest a physiological role for amylin in the regulation of tracheal mucus secretion.

Acetylcholine↗
PubMed1993

Partial hepatectomy affects pancreatic size and function in rats.

The effect of partial hepatectomy (62 +/- 2% of liver mass) or sham laparotomy on the pancreas was studied in rats. Pancreatic contents of DNA, protein, and digestive enzymes were measured 14 days postoperatively. Pancreatic acini were prepared to study exocrine pancreatic function after hormonal stimulation. Islet hormone release was investigated in the isolated perfused pancreas. Liver regeneration reached 93 +/- 1% within 14 days. Water contents in liver and pancreas remained unaltered. Simultaneously, the pancreatic weight increased significantly. Pancreatic enzymes showed a parallel elevation, whereas DNA remained unaffected. Amylase secretion from pancreatic acini was unaltered. Stimulated insulin and somatostatin release from the perfused pancreas were both increased. Plasma cholecystokinin levels were elevated, whereas neurotensin was decreased. Insulin and gastrin remained unchanged. In conclusion, after partial hepatectomy, enhanced cholecystokinin and decreased neurotensin blood levels are suggested to contribute to a hypertrophic effect on the exocrine pancreas and an adaptive regulation of the endocrine gland.

Amylases↗
PubMed1992

Expression of the ras-related rab3a gene in insulinoma-derived cell lines.

This study was designed to search for the expression of the small-molecular-weight GTP-binding protein rab3a in endocrine pancreatic cell lines. Total RNA was isolated from five different cell lines (RINm5F, RIN 104836, beta-TC1, HIT-15, and INRI-G9) and from whole rat brain. The expression of rab3a was analyzed by Northern blots. Similar as in brain two transcripts of 1300 and 1800 bp were detected in RIN-cells at low stringency conditions with the predominant signal at 1300 bp. At high stringency the stronger signal was at 1800 bp. When a 300 bp PstI fragment derived from the coding region of rab3a was utilized as probe the 1800 bp signal was predominant under each condition. Only a faint band at 1800 bp occurred in preparations from beta TC1-cells and no signal at all was found in HIT-15 and INRI-G9-cells. In conclusion, rab3a is expressed in rat insulin-releasing insulinoma-derived RIN-cells with a specific 1800 bp transcription product.

Animals↗
PubMed1992

Characterisation of the expression and post-translational processing of the preprotachykinin-I gene and the regulated release of tachykinins by the RINm5F cell-line.

Normal transcription and postranslational processing of the preprotachykinin (PPT)-I gene and regulated release of substance P and neurokinin A by the rat pancreatic endocrine cell-line, RINm5F, has been demonstrated, using radioimmunoassays (RIAs), reversed-phase (rp)HPLC and Northern blot analysis. This is the first stable cell-line found to express the PPT-I gene and provides an opportunity for investigating PPT-I gene expression and tachykinin biosynthesis. RIN5mF cells are a model for the pancreatic beta-cell, which is not known to exhibit PPT-I gene expression which may, therefore, be a feature of the transformed state of these cells. These data may imply that the tachykinins are important in pancreatic islet embryogenesis.

Animals↗
PubMed1992

Glucagon-like peptide-1(7-37)/(7-36)amide is a new incretin.

Glucagon-like peptide-1 (GLP-1) is the main product of the intestinal processing of proglucagon. It is released from the intestinal K-cells into the circulation in response to the oral ingestion of food. At the pancreatic beta cell GLP-1 is a potent insulin secretagogue in the presence of elevated glucose levels, defining glucagon-like peptide-1 as a new incretin. Its action is mediated by specific receptors coupled to the adenylate cyclase system by a stimulatory G-protein. Finally, glucagon-like peptide-1 stimulates proinsulin gene expression and it is thus involved at several levels in the regulation of insulin synthesis and secretion.

Adenylyl Cyclases↗
PubMed1992

Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells.

Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal peptide hormone that is released in response to oral nutrients and that potently augments glucose-mediated insulin secretion. GLP-I(7-37) has potent insulin-releasing activities in vivo in response to oral nutrients, in situ in the isolated perfused pancreas, and in vitro in cultured pancreatic B-cells. As such GLP-I(7-37) is a potent hormonal mediator in the enteroinsular axis involved in the regulation of glucose homeostasis. We now show that in addition to stimulating the release of insulin, GLP-I(7-37) stimulates proinsulin gene expression at the levels of gene transcription and cellular levels of proinsulin messenger RNA as well as the translational biosynthesis of proinsulin. These findings of the positive anabolic actions of GLP-I(7-37) on the synthesis of insulin in B-cells support the notion that GLP-I(7-37) may be of therapeutic use in stimulating the production of insulin in patients with noninsulin-dependent diabetes mellitus and that overproduction of insulin with subsequent hypoglycemia will not occur in response to the administration of GLP-I(7-37). Furthermore, these positive actions of GLP-I(7-37) on insulin production obviate the possibility of B-cell exhaustion in response to such a potent secretagogue.

Animals↗