Biomedical subjects

B Lankat-Buttgereit

Publications and source records attributed to B Lankat-Buttgereit.

At least 19 recordsLinked to original sources

PubMed2000

Cloning and quantitative determination of the human Ca2+/calmodulin-dependent protein kinase II (CaMK II) isoforms in human beta cells.

AIMS/HYPOTHESIS: The Ca2+/calmodulin-dependent protein kinase II (CaMK II) is highly expressed in pancreatic islets and associated with insulin secretion vesicles. The suppression of CaMK II disturbs insulin secretion and insulin gene expression. There are four isoforms of CaMK II, alpha to delta, that are expressed from different genes in mammals. Our aim was to identify the isoforms of CaMK II expressed in human beta cells by molecular cloning from a human insulinoma cDNA library and to assess its distribution in humans. METHODS: The previously unknown complete coding sequences of human CaMK IIbeta and the kinase domain of CaMK IIdelta were cloned from a human insulinoma cDNA library. Quantitative determination of CaMK II isoform mRNA was carried out in several tissues and beta cells purified by fluorescence activated cell sorting and compared to the housekeeping enzyme pyruvate dehydrogenase. RESULTS: We found CaMK IIbeta occurred in three splice variants and was highly expressed in endocrine tissues such as adrenals, pituitary and beta cells. Liver showed moderate expression but adipose tissue or lymphocytes had very low levels of CaMK IIbeta-mRNA. In human beta cells CaMK IIbeta and delta were expressed equally with pyruvate dehydrogenase whereas tenfold lower expression of CaMK IIgamma and no expression of CaMK IIalpha were found. CONCLUSION/INTERPRETATION: Although CaMK IIdelta is ubiquitously expressed, CaMK IIbeta shows preferential expression in neuroendocrine tissues. In comparison with the expression of a key regulatory enzyme in glucose oxidation, pyruvate dehydrogenase, two of the four CaM kinases investigated are expressed at equally high levels, which supports an important role in beta-cell physiology. These results provide the basis for exploring the pathophysiological relevance of CaMK IIbeta in human diabetes.

Adipose Tissue↗

PubMed2000

A novel silencer element repressing expression of the GLP-1 receptor gene in fibroblasts and pancreatic A-cells, but not in pancreatic B- and D-cells.

The effects of the incretin hormone glucagon-like peptide 1 (7-36)amide (GLP-1) are mediated by the GLP-1 receptor (GLP-1R). This is expressed in a cell- and tissue-specific manner. Recently, we have cloned the 5'-flanking region of the human GLP-1R gene. The basal promoter activity is driven by the ubiquitous transcription factor Sp1. The tissue- and cell-specific expression of the gene requires several negatively acting cis-regulatory elements. We have now characterized one so far unknown distal cell-specific silencer element (DCS), repressing gene transcription of the human GLP-1R gene in fibroblasts and pancreatic A-cells, but not in pancreatic B- and D-cells. Our data suggests that the basal activity of the GLP-1R promoter is repressed in a tissue- and cell-specific manner by this novel silencer element.

Animals↗

PubMed1999

The transporter associated with antigen processing TAP: structure and function.

The transport of antigenic peptides from the cytosol to the lumen of the endoplasmic reticulum (ER) is an essential process for presentation to cytotoxic T-lymphocytes. The transporter associated with antigen processing (TAP) is responsible for the intracellular translocation of peptides across the membrane of the ER. Efficient assembly of MHC-peptide complex requires the formation of a macromolecular transport and chaperone complex composed of TAP, tapasin and MHC class I molecules. Therefore, structure and function of TAP is important for the understanding of the immune surveillance.

Antigens↗

PubMed1999

Gene expression of the human glucagon-like peptide-1 receptor is regulated by Sp1 and Sp3.

The human glucagon-like peptide-1 (GLP-1) receptor mediates the insulinotropic effects of the incretin hormone GLP-1. It is expressed in a cell- and tissue-specific manner. Recently, we cloned the 5'-region of the GLP-1 receptor gene and found that tissue and cell specificity is lost by 5'-deletion to -574. In this region proximal to the main transcription start point three putative binding sites for Sp1 were localized. Now, in vitro binding of Sp1 was shown by deoxyribonuclease footprint analysis with DNA fragments using either recombinant Sp1 or nuclear extracts from HIT cells. To elucidate the roles of the three Sp1-binding sites, we mutated each of the sites individually as well as in different combinations. The activity of each construct was analyzed in comparison to the wild-type promoter. Mutation of two adjacent Sp1-binding sites showed a clear reduction of activity. Contrasting results were obtained after mutation of the third, more distal Sp1-binding site. Here, a clear increase (approximately 150%) revealed a silencing effect of this cis-regulatory element, possibly resembling a Sp3-binding site. Electrophoretic mobility shift analysis revealed binding of Sp1 and Sp3, which was demonstrated by supershifts using specific antibodies. Cotransfection with Sp1 and Sp3 expression vectors in insect cells lacking endogenous Sp factors clearly demonstrated the involvement of Sp1 and Sp3. Therefore, the basal activity of the GLP-1 receptor gene is mediated by two proximal Sp1-binding sites, whereas a more distal site acts as a repressor.

Animals↗

PubMed1998

Contribution of a PS1-like element to the tissue- and cell-specific expression of the human GLP-1 receptor gene.

The GLP-1 receptor (GLP-1R) mediates the insulinotropic effects of the incretion hormone glucagon-like peptide 1 (7-36) amide (GLP-1). Recently, we cloned the 5'-flanking region of the human GLP-1R gene. To characterize tissue- and cell-specific cis-regulatory elements, we constructed a series of 5'-deletions of the promoter. The activity of these constructs was tested in different cell lines. An element with high homology to PS1 was found to repress GLP-1R promoter activity in fibroblasts and pancreatic D-cells, but was not active in pancreatic A- and B-cells. PS1 was described to inhibit activation of a D-cell-specific enhancer. Cloning the PS1-like element upstream a heterologous promoter (SV40) revealed that it is functionally active independently from this enhancer. Our data suggest that basal activity of the GLP-1R promoter is silenced in a tissue- and cell-specific manner by negatively acting cis-regulatory elements, including a PS1-like element.

Animals↗

PubMed1998

The genomic organization of the human GLP-1 receptor gene.

The genomic organization of the human gene encoding the receptor for glucagon-like peptide-1 (GLP-1 (7-37)/(7-36) amide) was analyzed to reveal the relationship to other G-protein-coupled receptors. The coding sequence of the GLP-1 receptor is interrupted by 12 introns. These introns are uniformly distributed within the open reading frame. The length of the introns varies between 6.6 kb and 100 bp, in contrast to the relative constant length of 100 bp of the exons. All of the exon/intron splice junctions characterized followed the consensus GT-AG rule. A comparison of the genomic structure with other related receptor genes indicates that the exon/intron organization is well-conserved among the VIP/ glucagon/secretin receptor family.

3' Untranslated Regions↗

PubMed1997

Cloning and characterization of the 5' flanking sequences (promoter region) of the human GLP-1 receptor gene.

The glucagon-like peptide 1 (7-36) amide (GLP-1) receptor mediates the insulinotropic effects of the incretin hormone GLP-1. To elucidate the tissue-specific regulation of the GLP-1 receptor we screened a human genomic library with a human GLP-1 receptor cDNA. The gene spans 40 kb and consists of at least seven exons. The promoter contained no TATA- or CAAT-boxes, but several other putative cis-regulatory recognition sequences including three Sp1 binding sites. Transient transfections of GLP-1 receptor producing and non-producing cells with promoter/ reporter gene constructs revealed that the putative Sp1 binding sites and several other silencer and tissue specific elements are important for the activity. Therefore, 3000 bp upstream the GLP-1 receptor coding sequences comprise regulatory elements essential for the tissue- and cell-specific transcription of the gene.

Base Sequence↗

PubMed1995

Molecular cloning, functional expression, and signal transduction of the GIP-receptor cloned from a human insulinoma.

Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in the regulation of postprandial insulin secretion and proinsulin gene expression of pancreatic beta-cells. This study demonstrates the molecular cloning of a cDNA for the GIP-receptor from a human insulinoma lambda gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 466 amino acids which showed high homology (41%) to the human glucagon-like peptide 1 (GLP-1) receptor. Homology to the GIP receptor from rat or hamster was 79% and 81%, respectively. When transfected stably into fibroblast CHL-cells a high affinity receptor was expressed which coupled to the adenylate cyclase with normal basal cAMP and increasing intracellular cAMP levels under stimulation with human GIP-1-42 (EC50 = 1.29 x 10(-13) M). The receptor accepted only human GIP 1-42 (Kd = 1.93 +/- 0.2 x 10(-8) M) and porcine truncated GIP 1-30 (Kd = 1.13 +/- 0.1 x 10(-8) M) as high affinity ligands. At 1 microM, exendin-4 and (9-39)amide weakly reduced GIP-binding (25%) whereas secretin, glucagon, glucagon-like peptide-1, vasoactive intestinal polypeptide, peptide histidine-isoleucine, and pituitary adenylyl cyclase activating peptide were without effect. In transfected CHL cells, GIP-1-42 did not increase intracellular calcium. Northern analysis revealed one transcript of human GIP receptor mRNA with an apparent size of 5.5 kb. The exact understanding of GIP receptor regulation and signal transduction will aid in the understanding of the incretin hormone's failure to exert its biological action at the pancreatic B-cell in type II diabetes mellitus.

Amino Acid Sequence↗

PubMed1994

Signal transduction of the GLP-1-receptor cloned from a human insulinoma.

GLP-1 (glucagon-like peptide 1 (7-36) amide) plays an important role in the regulation of insulin secretion and proinsulin gene expression of pancreatic beta-cells. Patients with insulinoma tumors show uncontrolled insulin hypersecretion. This study demonstrates the molecular cloning of a cDNA for the GLP-1 receptor from a human insulinoma employing a lambda-gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 463 amino acids which showed high homology to the GLP-1 receptor in normal human pancreas. Four amino acid exchanges were found in comparison to a receptor sequence obtained from regular pancreatic islets. When transfected transiently into COS-7 or stably into fibroblast CHL cells a high affinity receptor was expressed which coupled to the adenylate cyclase with normal basal cAMP and increasing intracellular cAMP levels under GLP-1 stimulation. The receptor accepted GLP-1 and the non-mammalian agonist exendin-4 as high affinity ligands. In transfected COS-7 cells, GLP-1 did not influence intracellular calcium, whereas in the stably transfected fibroblasts GLP-1 transiently increased intracellular calcium to a small extent. The understanding of GLP-1 receptor regulation and signal transduction will aid in the discovery of compounds that act as agonists of the GLP-1 receptor for potential use in the treatment of diabetes and will facilitate the understanding of its expression under normal and pathophysiological conditions.

Amino Acid Sequence↗

PubMed1994

Glycosylation of the GLP-1 receptor is a prerequisite for regular receptor function.

The GLP-1 receptor on RINm5F cells is a glycoprotein with a M(r) of 63,000. Treatment of the receptor with glycopeptidase F generated a protein with a M(r) of 51,000, indicating that the GLP-1 receptor contains N-linked glycans. Tunicamycin pretreatment concentration-dependently decreased GLP-1 binding to RINm5F cells due to a decreased receptor number without change of receptor affinity. Tunicamycin exerted no effect on the GLP-1 receptor mRNA expression. The stimulation of cAMP production was decreased in tunicamycin-treated cells. Our data show that glycosylation of the GLP-1 receptor is a precondition for regular receptor function.

Amidohydrolases↗

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

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↗

PubMed1993

Solubilization of active receptors for glucagon-like peptide-1(7-36)amide from rat lung membranes.

We report on a protocol that allows the solubilization of active glucagon-like peptide (GLP)-1-(7-36)amide receptors from rat lung membranes. Digitonin-solubilized GLP-1(7-36)amide binding proteins from lung membranes most effectively, whereas (3-[(3-cholamidopropyl)- dimethylamino]-1-propane-sulfonate was less affective, and octyl-beta-glucoside, Triton X-100 and Lubrol PX were almost ineffective. Solubilization of binding activity was optimal at a digitonin concentration of 1%, a protein-to-detergent ratio of 1:10, and a pH between 7.0 and 8.0. Binding of GLP-1(7-36)amide to solubilized receptors was dependent on the concentration of solubilized protein. The presence of certain mono- and divalent cations was crucial for binding of GLP-1(7-36)amide to solubilized receptors. Scatchard analysis of the binding data revealed a single class of binding sites with dissociation and maximum binding constant values of 0.40 +/- 0.20 nM and 80.0 +/- 26.0 fmol/mg protein for membrane bound and 7.0 +/- 0.6 microM and 12.0 +/- 6.0 nmol/mg protein for solubilized receptors, respectively. In cross-linking experiments 125I-labeled GLP-1(7-36)amide was covalently attached to GLP-1(7-36)amide receptors on lung membranes. The apparent molecular mass of the solubilized receptor was 55,000 Da. This was proven in another experiment when receptor was consecutively cross-linked after solubilization. Nonhydrolyzable GTP analogues (GTP gamma S or GDP beta S) were unable to reduce GLP-1(7-36)amide-binding at solubilized receptors. This argues that the receptor is solubilized as a single protein and not as a receptor-G protein complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

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

Effects of norepinephrine on the oxidative pentose phosphate pathway in the rat heart.

To examine whether stimulation of alpha-adrenergic receptors may affect the oxidative pentose phosphate pathway (PPP) in the rat heart, norepinephrine (NE) and the alpha-adrenergic agonist norfenephrine were used. NE was administered as a continuous intravenous infusion in awake rats for 3 days. It stimulated the activity of cardiac glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of the oxidative PPP, in a dose-dependent manner. With the highest dose (0.2 mg.kg-1.hr-1), there was also a time-dependent enhancement. The increase observed after 48 hours was attenuated partially by the beta-receptor blocker metoprolol and the alpha-receptor blocker prazosin. It was entirely abolished when both drugs were administered. Carvedilol, a beta-adrenergic blocker and vasodilator with alpha 1-blocking activity (0.5 mg.kg-1.hr-1), prevented the NE-induced increase in cardiac G-6-PD activity, in functional parameters (heart rate, left ventricular systolic pressure, and left ventricular dP/dtmax), and in the heart weight/body weight ratio. The alpha-adrenergic stimulator norfenephrine increased myocardial G-6-PD activity; prazosin prevented this stimulation. NE and norfenephrine also elevated the available pool of cardiac 5-phosphoribosyl-1-pyrophosphate. G-6-PD activity was enhanced in cardiac myocytes freshly isolated from the left ventricle of rats that had received NE infusion for 3 days (12.3 +/- 1.4 units/g protein) compared with control rats (1.5 +/- 0.4 units/g protein). The activity of 6-phosphogluconate dehydrogenase, one of the enzymes in the oxidative PPP, was elevated only moderately from 12.7 +/- 0.7 to 19.1 +/- 1.4 units/g protein. Combined alpha- and beta-receptor blockade with carvedilol attenuated these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗