Rüdiger Göke

Glucagon-Like Peptide-1 and Glucose-Dependent Insulin-Releasing Polypeptide Plasma Levels in Response to Nutrients

The nutrient-dependent glucagon-like peptide-1 (7-36) amide (GLP-1) release was studied in comparison to the glucose-dependent insulin-releasing polypeptide (GIP) response in 10 healthy volunteers each undergoing various protocols. Plasma samples were saved up to 120 min after challenges by oral, intravenous or intraduodenal administration of nutrients. Basal plasma-GLP-1 concentrations ranged between 0.4 and 1.4 pM, maximal postprandial GLP-1 levels peaked between 10 and 12 pM. Intravenous glucose (25 g i.v.) did not change basal GLP-1 levels. Oral administration of glucose (50 g) induced a biphasic GLP-1 release peaking at 30-60 min and a biphasic GIP release peaking at 5 and 45 min. This increase paralleled the secretion of insulin. Oral galactose (100 g) and amino acids (25 g) also induced a rapid plasma GLP-1 response. After fat (67 g corn oil) a strong and long-lasting (> 120 min) increase of GLP-1 plasma levels occurred. When a mixed liquid meal was given (6 g soybean oil, 5 g casein, 13 g glucose) immunoreactive (IR)-GLP-1 rapidly increased and peaked after 5 min with declining levels after 30 min. In response to an intraduodenal infusion of a small glucose load (5.34 g within 120 min) a rapid, short-lasting GLP-1 response occurred whereas plasma GIP and insulin levels remained unaltered. Luminal perfusion of an isolated vascularly perfused rat ileum with a polydiet induced a rapid rise of portally released IR-GLP-1 which was followed by a sustained release. Glucose evoked sodium-dependently a sharp increase of IR-GLP-1 levels followed by a plateau release. The intraluminal infusion of a mixture of amino acids or fat was without any effect on IR-GLP-1. We hypothesize that in contrast to GIP the GLP-1 release from L cells is triggered by nervous reflexes, by putative humoral factor(s) being released from the upper small intestine in addition to nutrient stimuli acting at the luminal surface of the gut.

The isolated N-terminal extracellular domain of the glucagon-like peptide-1 (GLP)-1 receptor has intrinsic binding activity

The glucagon‐like peptide 1 (7–37)/(7–36) amide (GLP‐1) receptor belongs to a new subclass of seven transmembrane domain, G‐protein coupled receptors comprising several receptors for peptide hormones. The receptors of this family share many common motifs including a relatively large N‐terminal extracellular domain. The GLP‐1 receptor is presently attracting much attention, since it is the target protein of the antidiabetic gut hormone GLP‐1. To establish the functional significance of the N‐terminal part of the GLP‐1 receptor for ligand binding, the extracellular domain was isolated and purified. Utilizing CHL cells expressing the cloned GLP‐1 receptor, we demonstrate that the isolated, solubilized N‐terminal part of the receptor protein competes for GLP‐1 binding with the intact wild‐type receptor. Moreover, in cross‐linking experiments radiolabeled GLP‐1 was covalently attached to the isolated N‐terminus, thereby demonstrating direct physical interaction of both components. By Western blot analysis two specific bands were detectable, representing the N‐terminal receptor protein in the presence or absence of bound ligand. These data underline the significance of the N‐terminal domain of the GLP‐1 receptor for ligand binding.

Glucagon-like peptide-1 cells in the gastrointestinal tract and pancreas of rat, pig and man

Abstract. A highly specific monoclonal antibody directed against the C‐terminal part of glucagon‐like peptide‐1 (GLP‐1) was raised to immunohistochemi‐cally evaluate the distribution of GLP‐1 containing cells in the entire gastrointestinal tract including pancreas of rat, pig and man. In the pancreas GLP‐1 ‐immunoreactive cells were found variously shaped and predominantly located in the periphery of the islets. Ultrastructurally, GLP‐1 was co‐localized with gluca‐gon in the α‐granula of A‐cells and was mainly restricted to the electrondense core. In the intestine open type cells reaching the lumen via a slender apical process were stained with the GLP‐1 antibody. They occurred in all parts of the crypts but predominantly in the basal portion. The density of GLP‐1 immuno‐reactive cells varied between species in a characteristic order: rat > pig > man. In pig and human gut a large number of cells occurred in the distal jejunum and ileum. A continuous increase of cell densities was found from the proximal to the distal colon resulting in highest numbers in the rectum. In rats the highest cell density occurred in the ileum. Again, a continuous increase of GLP‐1‐positive cell numbers was evident from the proximal to the distal portion of small and large bowel. GLP‐1 was partly co‐localized with PYY. The GLP‐1 positive cells appeared electronmicrosco‐pically as L‐cells with the typical large granula. This morphological data indicates that GLP‐1‐releasing cells in the small intestine are appropriately positioned in the distal part to sense and respond to the presence of nutrients that have escaped the absorptive surface of the upper small intestine.

Distribution of GLP‐1 Binding Sites in the Rat Brain: Evidence that Exendin‐4 is a Ligand of Brain GLP‐1 Binding Sites

The distribution and biochemical properties of glucagon‐like peptide (GLP)‐1(7–36)amide (GLP‐1) binding sites in the rat brain were investigated. By receptor autoradiography of tissue sections, the highest densities of [125I]GLP‐1 binding sites were identified in the lateral septum, the subfornical organ (SFO), the thalamus, the hypothalamus, the interpenduncular nucleus, the posterodorsal tegmental nucleus, the area postrema (AP), the inferior olive and the nucleus of the solitary tract (NTS). Binding studies with [125I][Tyr39]exendin‐4, a GLP‐1 receptor agonist, showed an identical distribution pattern of binding sites. Binding specificity and affinity was investigated using sections of the brainstem containing the NTS. Binding of [125I]GLP‐1 to the NTS was inhibited concentration‐dependently by unlabelled GLP‐1 and [Tyr39]exendin‐4 with K1 values of 3.5 and 9.4 nM respectively. Cross‐linking of hypothalamic membranes with [125I]GLP‐1 or [125I][Tyr39]exendin‐4 identified a single ligand‐binding protein complex with a molecular mass of 63 000 Da. The fact that no GLP‐1 binding sites were detected in the cortex but that they were detected in the phylogenetically oldest parts of the brain emphasizes that GLP‐1 may be involved in the regulation of vital functions. In conclusion, the biochemical data support the idea that the central GLP‐1 receptor resembles the peripheral GLP‐1 receptor. Furthermore, the presence of GLP‐1 binding sites in the circumventricular organs suggests that these may be receptors which act as the target for both peripheral blood‐borne GLP‐1 and GLP‐1 in the nervous system.

The tumour suppressor Pdcd4: recent advances in the elucidation of function and regulation

Pdcd4 (programmed cell death 4) has been known as a tumour suppressor gene and potential target for anticancer therapies for several years. Initially, Pdcd4 was identified as a gene that is up‐regulated during apoptosis, but its precise role still remains to be defined. However, there is increasing evidence that Pdcd4 levels influence transcription, as well as translation, modulate different signal transduction pathways and might act as a tumour suppressor. Interestingly, recent data suggest that Pdcd4 function may depend on cell type and/or genetic background. This review summarizes the current knowledge regarding the function and regulation of Pdcd4.

Reduction of the Incretin Effect in Rats by the Glucagon-Like Peptide 1 Receptor Antagonist Exendin (9–39) Amide

Glucagon-like peptide 1 (7–37)/(7–36) amide (GLP-1) is derived from the intestinal proglucagon processing. It is considered an important insulin-releasing gut hormone. This study uses exendin (9–39) amide as a GLP-1 receptor antagonist to evaluate the contribution of GLP-1 to the incretin effect. Anesthetized rats were challenged by an intraduodenal glucose infusion to evaluate maximally occurring GLP-1 and gastric inhibitory polypeptide (GIP) plasma levels. Maximal immunoreactive (IR) GLP-1 plasma levels amounted to 10 pmol/l (IR-GIP 11 pmol/l). Exendin (9–39) amide abolished the insulin-stimulatory effect of 60 pmol of GLP-1 or of the GLP-1 agonist exendin-4 (0.5 nmol) injected as bolus, respectively. An intravenous bolus injection of 5.94 nmol of exendin (9–39) amide 3 min before enteral glucose infusion grossly reduced the total insulin secretory response (by 60%) and significantly increased circulating blood glucose levels (P < 0.05). In contrast, the GLP-1 antagonist left the insulin response after an intravenous glucose or glucose plus GIP (60 pmol) load unaltered. Our data support the concept that GLP-1 is an important incretin factor. Exendin (9–39) amide is a useful GLP-1 antagonist for in vivo studies.

Roles of TNF-Related Apoptosis-Inducing Ligand in Experimental Autoimmune Encephalomyelitis

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL receptor, we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were significantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells.

Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides.

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.

Translational Regulation of Autoimmune Inflammation and Lymphoma Genesis by Programmed Cell Death 4

Both inflammatory diseases and cancer are associated with heightened protein translation. However, the mechanisms of translational regulation and the roles of translation factors in these diseases are not clear. Programmed cell death 4 (PDCD4) is a newly described inhibitor of protein translation. To determine the roles of PDCD4 in vivo, we generated PDCD4-deficient mice by gene targeting. We report here that mice deficient in PDCD4 develop spontaneous lymphomas and have a significantly reduced life span. Most tumors are of the B lymphoid origin with frequent metastasis to liver and kidney. However, PDCD4-deficient mice are resistant to inflammatory diseases such as autoimmune encephalomyelitis and diabetes. Mechanistic studies reveal that upon activation, PDCD4-deficient lymphocytes preferentially produce cytokines that promote oncogenesis but inhibit inflammation. These results establish that PDCD4 controls lymphoma genesis and autoimmune inflammation by selectively inhibiting protein translation in the immune system.

Glucagon-like peptide-1(7–36) amide is a new incretin/enterogastrone candidate

Cloning and sequence analysis of cDNAs and DNA fragments from genomic libraries has led to a dramatic increase in understanding of the glucagon-related peptides in recent years. The primary structure of the biosynthetic precursor of glucagon (preproglucagon) has been elucidated. The complex structural connec- tions between the multiple molecular forms of the glucagon-like peptides in tissues and in the circulation have been determined [ 1,2]. Mammalian proglucagon consists of 160 amino acid residues and is synthesized in the islets of Langerhans, intestine and brain. An exciting recent finding is that in addition to glucagon the preproglucagon gene in mammals encodes two additional peptides with struc- tural similarity to glucagon, termed glucagon-like peptide-1 and -2 (GLP-I and GLP-2). The truncated form of GLP-1, GLP-1(7-36) amide, which is secreted by the mammalian intestine, strongly stimulates insu- lin secretion and inhibits gastric acid secretion. This review focuses mainly on the truncated form of GLP-1. since a rapidly increasing number of reports indicate a remarkable interest of investigators in this particular hormone, which actually fulfills the classical role of an ‘enterogastrone’ and ‘incretin’ hormone. The purpose here is to describe what is known so far about the origin, processing, organ distribution and actions of this peptide and to search for promising future direc- tions of further investigations.