Søren P. Sheikh

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.

Y1 and Y2 receptors for neuropeptide Y

By using monoiodinated radioligands of both intact neuropeptide Y (NPY) and of a long C‐terminal fragment, NPY13–36, two subtypes of binding sites, which differ in affinity and specificity, have been characterized. The Y1 type of binding site, characterized on a human neuroblastoma cell line, MC‐IXC, and a rat pheochromocytoma cell line, PC‐12, binds NPY with a dissociation constant (K d) of a few nanomolar but does not bind NPY13–36. The Y2 type of binding site, characterized on porcine hippocampal membranes and on another human neuroblastoma cell line, SMS‐MSN, is of higher affinity and binds both NPY and NPY13–36. None of the binding sites distinguish between NPY and the homologous peptide YY (PYY). It is concluded that NPY/PYY‐binding sites occur in two subtypes which may represent two types of physiological receptors.

Regional Distribution of Putative NPY Y1 Receptors and Neurons Expressing Y1 mRNA in Forebrain Areas of the Rat Central Nervous System

Using monoiodinated radioligands of peptide YY (PYY), and the recently introduced neuropeptide Y (NPY) analogue [Leu31,Pro34]NPY, receptor binding sites of the Y1 and Y2 type were localized in the rat brain by quantitative in vitro autoradiography. The binding specificity and affinity of both radiolabeled ligands were analysed by ligand binding studies employing rat brain membrane homogenates from cerebral cortex and hippocampus. Using in situ hybridization histochemistry, the regional distribution and cellular localization of mRNA encoding the Y1 receptor were investigated in rat brain sections and compared to the distribution of Y1‐specific binding sites. PYY binds to both Y1 and Y2 receptors, while long C‐terminal fragments such as NPY13–36 and NPY16–36 bind preferentially to Y2 receptors. [Leu31, Pro34]NPY is a specific agonist for Y1 receptors. Highest densities of [125I]PYY binding sites were found in the cerebral cortex, the thalamus, the lateral septum, the hippocampus and the mesencephalic dopaminergic areas. In order to block putative Y2 receptors, a series of [125I]PYY binding experiments was performed in the presence of NPY13–36 (1 μM), a Y2 preferring C‐terminal fragment. High densities of binding sites remained present in the cerebral cortex, the thalamus and the medial mammillary nucleus when NPY13–36 was present in the incubation medium. Furthermore, these areas were highly enriched with [125l][Leu31, Pro34]NPY binding sites. In contrast, the hippocampal complex had its binding capacity reduced by ‐50%, while the lateral septum and mesencephalic dopaminergic areas had their binding capacities reduced even further. Linear regression analysis showed a high degree of correspondence between [125l][Leu31, Pro34]NPY binding and that obtained with [125I]PYY in the presence of 1 μM NPY13–36, suggesting that the two independent approaches to visualizing Y1 binding sites are comparable. In situ hybridization histochemistry revealed high levels of Y1 mRNA in the granular cell layer of the hippocampal dentate gyrus, several thalamic nuclei and the hypothalamic arcuate nucleus. Moderate levels of Y1 mRNA were seen in the frontoparietal cortex, several thalamic nuclei, the hippocampal pyramidal layers, the subiculum, the olfactory tubercle, the claustrum and a number of hypothalamic nuclei. The mesencephalon, the amygdala and most basal ganglia showed very low levels of hybridization. The present study further clarifies the anatomical distribution of multiple NPY binding sites within the central nervous system of the rat, and extends earlier suggestions that Y1 and Y2 receptor types are present within the central nervous system.

Glucagon-like peptide-1 7-36 amide and peptide YY from the L-cell of the ileal mucosa are potent inhibitors of vagally induced gastric acid secretion in man

BACKGROUND Glucagon-like peptide (GLP-1) 7-36 amide and peptide YY (PYY) from the L-cell of the ileal mucosa are potent inhibitors of gastric acid secretion in man. It is not clear, however, by which mechanism(s) they inhibit acid secretion. In dogs the inhibitory effect of PYY on acid secretion may be mediated mainly through neural pathways. The mechanism of action of GLP-1 might be similar. The aim of the present study was to examine the effects of GLP-1 might be similar. The aim of the present study was to examine the effects of GLP-1 and PYY on the vagally induced gastric acid secretion in man. METHODS A modified sham feeding technique, chew and spit, was used. Six healthy volunteers were randomly assigned to receive intravenous infusion of saline, GLP-1 (41 pmol/kg/h), or peptide YY (50 pmol/kg/h). RESULTS The infusion of GLP-1 and PYY resulted in plasma concentrations of 60 +/- 9 pmol/l and 84 +/- 11 pmol/l, respectively. GLP-1 and PYY both significantly inhibited the intergrated acid output by 67 +/- 6% and 68 +/- 9%, respectively, compared with the integrated outputs in a control experiment with saline infusion. Serum gastrin and plasma somatostatin concentrations remained unchanged during saline, GLP-1, and PYY infusions. CONCLUSIONS GLP-1 and PYY are both potent inhibitors of the cephalic phase of acid secretion, indicating that at least part of the inhibitory effect of GLP-1 and PYY in man is mediated through neural pathways. Furthermore, the inhibitory effect seems to be independent of circulating concentrations of gastrin and somatostatin.

Expression profiling reveals differences in metabolic gene expression between exercise‐induced cardiac effects and maladaptive cardiac hypertrophy

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise‐induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise‐induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in β‐oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise‐induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise‐induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.

Receptors on phaeochromocytoma cells for two members of the PP‐fold family — NPY and PP

Pancreatic polypeptide (PP) and neuropeptide Y (NPY) belong to a family of regulatory peptides which hold a distinct tertiary structure, the PP‐fold, even in dilute aqueous solution. High‐affinity receptors, specific for both PP and NPY, are described on the rat phaeochromocytoma cell line, PC‐12. The binding of [125I‐Tyr36]PP to PC‐12 cells was inhibited by concentrations of unlabeled PP which correspond to physiological concentrations of the hormone, 10−11‐10−9 mol/1. The affinity of the receptor for the neuropeptide, NPY, was 102‐times lower than that of the PP receptor. C‐terminal fragments of both PP (PP24–36) and NPY (NPY13–36) were between 102 ‐ and 103‐times less potent in displacing the radiolabeled 36‐amino‐acid peptides from their respective receptors. It is concluded that PC‐12 cells are suited for structure‐function studies of the PP‐fold peptides and studies on the cellular events following cellular binding of PP‐fold peptides.

Identification of Specific Binding Sites for Glucagon-Like Peptide-1 on the Posterior Lobe of the Rat Pituitary

Glucagon-like peptide-1 (GLP-1) immunoreactivity has been found in autonomic and neuroendocrine brain regions, whereas only limited data are available regarding the characterization and localization of brain GLP-1 receptors. In the present study, using quantitative in vitro autoradiography, a high density of specific binding sites for GLP-1 was characterized on sections of the posterior pituitary lobe of the rat. Low specific binding of radiolabeled GLP-1 was found in the anterior lobe and no specific binding in the intermediate lobe. To examine the specificity of GLP-1 binding sites, sections of the posterior lobe were incubated with radiolabeled GLP-1 in the presence of various peptides. Radiolabeled [Tyr39]exendin-4, a specific GLP-1 agonist, bound to these receptor sites with the same affinity as GLP-1, while glucagon and vasoactive intestinal peptide (VIP) were unable to displace 125I-GLP-1. Both unlabeled exendin-4 and GLP-1 inhibited this binding with equally high affinity. Using 125I-[Tyr39]exendin-4 as radiolabel, the concentration of biding sites was found to be 7.8 +/- 0.4 fmol/mg tissue. Further analysis of the binding data from experiments with tissue slices revealed the presence of high and low affinity binding sites. In experiments with unlabeled [Tyr39]exendin-4, the KdS were 6.2 +/- 1.4 x 10(-12) and 9.3 +/- 1.5 x 10(-10) M, respectively, and in experiments with unlabeled GLP-1, 3.4 +/- 1.8 x 10(-12) and 5.9 +/- 1.5 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmotic regulation of neuropeptide Y and its binding sites in the magnocellular hypothalamo-neurohypophysial pathway

The magnocellular hypothalamo-neurohypophysial system is, via a release of vasopressin from nerve terminals in the neurohypophysis to the peripheral blood, centrally involved in the regulation of body salt and water homeostasis. Furthermore, it has been shown that expression of neuropeptides co-existing with vasopressin or oxytocin in magnocellular neurons is influenced by salt loading. We here report, that neuropeptide Y (NPY)-immunoreactivity, which is normally not observed in the magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei of rats becomes immunohistochemically detectable after salt loading. Using a double-immunohistochemical procedure on the same brain sections, it is shown that NPY is co-existing with either vasopressin or oxytocin in these neurons. Within the neurohypophysis of normal rats, a moderate number of predominantly fine calibered NPY-immunoreactive nerve fibers most often coursing along vessels is observed in addition to a low number of large peptidergic terminals. In salt-loaded rats, however, the number of NPY-immunoreactive neurohypophysial large nerve terminals in apposition to vascular lumina is drastically increased. By using quantitative receptor autoradiography, it is demonstrated that in salt-loaded animals, the number of neurohypophysial NPY binding sites is decreased to nearly undetectable levels (0.054 +/- 0.02 fmol/mg) compared to a very high density of binding sites in normal animals (1.151 +/- 0.15 fmol/mg). This raises evidence that NPY containing hypothalamo-neurohypophysial neurons as well as peripherally released NPY may be involved in the regulation of water homeostasis via NPY receptors in the neurohypophysis.

Regional Distribution of Neuropeptide Y and Its Receptor in the Porcine Central Nervous System

Abstract: The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200–1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50–200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained <50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0–5.87 pmol/mg of protein. Intermediate binding (0.5–1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but <0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.

Solubilization of active GLP-1 (7–36)amide receptors from RINm5F plasma membranes

Glucagon‐like peptide‐1 (7–36)amide (GLP‐1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP‐1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP‐1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl‐β‐glucoside and CHAPS; Triton X‐100 and Lubrol PX were ineffective. Binding of radiolabeled GLP‐1(7–36)amide to the solubilized receptor was inhibited conentration‐dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K a= 0.26 ± 0.03 nM and B max= 65.4 ± 21.24 fmol/mg of protein for the membrane‐bound receptor and K a= 22.54 ± 4.42 μM and B max= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono‐ and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine‐nucleotides, however neither GTP‐γ‐S nor GDP‐β‐S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G‐protein. In conclusion, the here presented protocol allows solubilization of the GLP‐1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein.