Stimulation of insulin release by repaglinide and glibenclamide involves both common and distinct processes.

The action of repaglinide, a novel insulin secretagogue, was compared with the sulfonylurea glibenclamide with regard to the hypoglycemic action in vivo, binding to betaTC-3 cells, insulin secretion from perifused mouse islets, and capacity to stimulate exocytosis by direct interaction with the secretory machinery in single voltage-clamped mouse beta-cells. Two binding sites were identified: a high-affinity repaglinide (KD = 3.6 nmol/l) site having lower affinity for glibenclamide (14.4 nmol/l) and one high-affinity glibenclamide (25 nmol/l) site having lower affinity for repaglinide (550 nmol/l). In contrast to glibenclamide, repaglinide (in concentrations as high as 5 micromol/l) lacked the ability to enhance exocytosis in voltage-clamped beta-cells. Repaglinide was more potent than glibenclamide in stimulating insulin release from perifused mouse islets (EC50 29 vs. 80 nmol/l). The greater potency of repaglinide in vitro was paralleled by similar actions in vivo. The ED50 values for the hypoglycemic action were determined to be 10.4 and 15.6 microg/kg after intravenous and oral administration, respectively. The corresponding values for glibenclamide were 70.3 microg/kg (intravenous) and 203.2 microg/kg (oral). Further, repaglinide (1 mg/kg p.o.) was effective (P < 0.001) as an insulin-releasing agent in a rat model (low-dose streptozotocin) of type 2 diabetes. These observations suggest that the insulinotropic actions of repaglinide and glibenclamide in vitro and in vivo are secondary to their binding to the high-affinity repaglinide site and that the insulinotropic action of repaglinide involves both distinct and common cellular mechanisms.

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase.

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.

Structure-function studies on neuropeptide Y and pancreatic polypeptide--evidence for two PP-fold receptors in vas deferens.

The biological effects of neuropeptide Y (NPY), rat pancreatic polypeptide (rPP), hybrid analogs of NPY and PP, and C-terminal fragments of NPY were studied in the field-stimulated rat vas deferens model. The results were correlated with peptide binding experiments in Y1 and PP receptor assays on rat PC-12 cells and Y2 receptors on porcine hippocampal membranes. NPY and rPP inhibited the electrically induced contractions in the vas deferens with an IC50 of 25 and 22 nM respectively. However, in contrast to NPY, rPP could not totally block muscle activity. The inhibitory action of the long C-terminal fragment of NPY, NPY-(19-36) and NPY-(11-36), indicated that NPY acts through a Y2 receptor in the vas deferens. The structural basis for the differential recognition of NPY and PP by Y2 receptors and partly also by PP receptors, could be defined with hybrid analogs of PP and NPY. The analogs, [Ile31,Gln34]PP and [Leu31,Pro33]NPY reacted in the vas deferens preparation in accordance with their relative potency in the Y2 and PP receptor assays. [Ile31,Gln34]PP, which bound to the Y2 receptor like NPY, was also able to block the part of the contractile response which was resistant to rPP. It is concluded that in the vas deferens, PP-fold peptides act through two types of receptors: Y2 and PP, and that residues in the C-terminal part of the molecules determine the differential recognition of the peptides by these receptor types.

[Pro34]neuropeptide Y selectively identifies postjunctional-mediated actions of neuropeptide Y in vivo in rats and dogs.

Anaesthetised rats and dogs were used to study the pre- and postjunctional actions of neuropeptide Y (NPY) and [Pro34]NPY simultaneously. Increases in arterial blood pressure indicated postjunctional actions and both NPY and [Pro34]NPY elicited these. Change in pulse interval evoked by stimulation of the cut peripheral end of the right vagus nerve, indicated prejunctional action of the peptides: NPY caused prolonged attenuation of vagal action in rats and dogs but [Pro34]NPY did not attenuate vagal action in rats or dogs.

Repaglinide, glibenclamide and glimepiride administration to normal and hereditarily diabetic rats.

Repaglinide (1 microg/g body wt), glibenclamide (10 microg/g) or glimepiride (10 microg/g) were administered orally to either fed or overnight fasted normal rats and hereditarily diabetic animals (GK rats). In both fed and starved normal rats, repaglinide provoked a greater and more rapid increase in plasma insulin concentration and an earlier fall in glycemia than those observed after administration of the hypoglycemic sulfonylureas. Likewise, in fed GK rats, the plasma insulin concentration was already increased by 30.0 +/- 1.6% 15 min after administration of repaglinide, whilst a sizeable insulinotropic action of the sulfonylureas was only recorded at much later times. Except for a lower glycemia at the 240th min of the test, there was little to distinguish, in starved GK rats, between control experiments including the oral administration of the solution of carboxymethylcellulose used as vehicle and the experiments conducted with the antidiabetic agents. Several converging observations indicated that glimepiride stimulated insulin release more promptly than glibenclamide. It is proposed that advantage can be taken from these vastly different time-courses of the hormonal and metabolic response to distinct hypoglycemic agents to optimize the control of glucose homeostasis in non-insulin-dependent diabetic subjects.

Primary structure of tetranectin, a plasminogen kringle 4 binding plasma protein: homology with asialoglycoprotein receptors and cartilage proteoglycan core protein.

Tetranectin binds to plasminogen and to isolated kringle 4 [Clemmensen, I., Petersen, L. C., & Kluft, C. (1986) Eur. J. Biochem. 156, 327-333], apparently to its lysine-binding site. Each of the four identical chains consists of 181 amino acid residues. The three intrachain disulfide bonds connect Cys residues 50-60, 77-176, and 152-168. The tetranectin sequence is homologous (17-24% identical positions) with those parts of the asialoglycoprotein receptor family that are considered to be extracellular. Tetranectin has no structures corresponding to those parts of the receptors considered to be intracellular and membrane anchoring. The sequence of tetranectin is also homologous (22-23% identical positions) with the C-terminal globular domain of the core protein of the cartilage proteoglycan. All six Cys residues in tetranectin are located at positions that are also Cys residues in this proteoglycan. Therefore, a plausible disulfide bond pattern can now be proposed for both the asialoglycoprotein receptors and the C-terminal domain of the proteoglycan core protein. No covalently bound carbohydrate has been found.

Effect of antidiabetic agents on the increase in glycemia and insulinemia caused by refeeding in hereditarily diabetic rats.

The hypoglycemic agents, glibenclamide and repaglinide, when administered intragastrically to overnight fasted hereditarily diabetic animals, were found to oppose the rise in the plasma insulin/glucose ratio otherwise evoked by refeeding of the GK rats. Such was not the case after oral administration of glimepiride, despite the fact that this sulfonylurea minimized the rise in glycemia associated with refeeding. The altered restoration of a high insulin/glucose ratio in GK rats that received glibenclamide or repaglinide before refeeding suggests that these long-acting hypoglycemic agents may delay the refeeding-induced relief of the B-cell from the fasting-associated refractoriness to glucose.

Localization of Y1 receptors for NPY and PYY on vascular smooth muscle cells in rat pancreas.

To localize binding sites for peptide YY (PYY) in the pancreas we utilized a slide-mount autoradiographic technique on frozen sections of rat pancreas incubated with 125I-Tyr36-PPY. Saturable autoradiographic labeling was located over pancreatic blood vessels, whereas islets, acinar cells, ducts, and neural elements did not appear to be specifically labeled. Isolated vascular fragments were prepared by collagenase digestion of rat pancreas. Binding experiments with 125I-Tyr36-PYY showed saturable binding to the fraction enriched in blood vessels but not to acini. Inhibition of 125I-Tyr36-PYY binding by nonradioactive neuropeptide Y (NPY) and PYY were similar, with half-maximal inhibition at 31.2 +/- 5 pM (n = 6); the potency of pancreatic polypeptide (PP) was 10,000 times lower. The binding site was classified as belonging to a Y1 type of NPY and/or PYY receptors, since [Leu31,Pro34]NPY, a specific Y1-receptor agonist, inhibited binding similar to NPY. To further localize the bound [125I-Tyr36]PYY within the blood vessels, light- and electron-microscopic autoradiographs were prepared and quantitated. Autoradiographic grains were located predominantly over vascular smooth muscle cells, although saturable localization was also seen over endothelial cells. It is concluded that in the pancreas Y1 receptors are predominantly located on vascular smooth muscle cells.

The antiparallel pancreatic polypeptide fold in the binding of neuropeptide Y to Y1 and Y2 receptors.

Neuropeptide Y (NPY) belongs to the pancreatic polypeptide fold (PP-fold) family of regulatory peptides. Analysis of circular dicroic spectra of NPY showed that it has a high degree of secondary structure in aqueous solution which is in agreement with the globular, folded crystal structure of PP. Using three different approaches with synthetic peptides, we have probed the importance of the PP-fold structure in the interaction of NPY with two types of binding sites, Y1 and Y2 receptors. First, stepwise construction of the NPY molecule from the C-terminal amidated end, showed that although C-terminal fragments encompassing most of the long alpha-helix reacted reasonably well with the Y2 receptor, both Y1 and Y2 receptors required the presence of both ends of the PP-fold for full activity. Second, perturbation of the PP-fold by substitution with a helix-breaking proline residue, resulted in the loss of recognition of the N-terminal segment of the molecule by both types of receptors. Finally, a hybrid analog was constructed in which the essential, but by itself inactive, C-terminal segment of NPY was joined with the PP-fold motif of PP. This segment of PP is only 43% homologous to the similar motif in NPY, and most of the common residues cluster in the hydrophobic core of the fold. Nevertheless, the hybrid analog reacted with almost full potency on the Y2 receptors. It is concluded that the antiparallel PP-fold is of structural importance for the receptor binding of NPY, and that its main function is to present the combined C- and N-terminal segments of the molecule to the receptors.

Stimulation of insulin and somatostatin release by two meglitinide analogs.

Several meglitinide analogs are currently under investigation as potential insulinotropic tools for the treatment of noninsulin-dependent diabetes. The present study aimed to further insight into the effect of these agents on the secretion of insulin, glucagon, and somatostatin by the isolated perfused pancreas. Both repaglinide (0.01 microM) and A-4166 (1.0 microM) stimulated insulin and somatostatin release, but failed to affect glucagon output, from pancreases exposed to 5.6 mM D-glucose. The secretory response of the B- and D-cells to the hypoglycemic agents was much less marked than that caused by a rise in hexose concentration from 5.6-16.7 mM. Although repaglinide was tested at a concentration a hundred times lower than that of A-4166, the drug-induced increase in both insulin and somatostatin secretion persisted for a longer time after exposure to repaglinide, than to A-4166. The relevance of these findings to the use of meglitinide analogs as antidiabetic agents is double. First, they document that these drugs, although enhancing both insulin and somatostatin release, do not provoke an undesirable stimulation of glucagon secretion. Second, they indicate that even at a very low concentration, repaglinide provokes a protracted insulinotropic action, thus suggesting that the reversibility of the secretory response to this or other meglitinide analogs represents an intrinsic molecular attribute, unrelated to either their biological potency or the relative extent of B-cell stimulation.

[Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist.

Two types of binding sites have previously been described for 36-amino acid neuropeptide Y (NPY), called Y1 and Y2 receptors. Y2 receptors can bind long C-terminal fragments of NPY-e.g., NPY-(13-36)-peptide. In contrast, Y1 receptors have until now only been characterized as NPY receptors that do not bind such fragments. In the present study an NPY analog is presented, [Leu31, Pro34]NPY, which in a series of human neuroblastoma cell lines and on rat PC-12 cells can displace radiolabeled NPY only from cells that express Y1 receptors and not from those expressing Y2 receptors. The radiolabeled analog, [125I-Tyr36] monoiodo-[Leu31, Pro34]NPY, also binds specifically only to cells with Y1 receptors. The binding of this analog to Y1 receptors on human neuroblastoma cells is associated with a transient increase in cytoplasmic free calcium concentrations similar to the response observed with NPY. [Leu31, Pro34]NPY is also active in vivo as it is even more potent than NPY in increasing blood pressure in anesthetized rats. It is concluded that [Leu31, Pro34]NPY is a specific Y1 receptor agonist and that the analog or variants of it can be useful in delineating the physiological importance of Y1 receptors.