A. Hasselblatt

An insulin-releasing property of imidazoline derivatives is not limited to compounds that block α-adrenoceptors

SummaryAs we have demonstrated previously phentolamine stimulates the release of additional insulin from isolated mouse islets and raises plasma insulin levels in the whole rat. This effect was independent of the well known property of phentolamine to block α-adrenoceptors.In experiments on isolated pancreatic islets from mice we now demonstrate that tolazoline and antazoline which are chemically closely related to phentolamine, share its ability to potentiate insulin release.The following results were taken as evidence that this effect does not result from an a-adrenoceptor blocking action of imidazoline compounds.More than 10 times higher concentrations of phentolamine were required to liberate additional insulin from isolated islets than were effective in counteracting the inhibitory effect of clonidine on insulin release.The newly introduced α2-adrenoceptor antagonist BDF 8933, which is an imidazoline derivative, stimulates insulin release as well, while the irreversible α-adrenoceptor blocking agent benextramine of different structure failed to do so, even when being present in concentrations blocking the α2-adrenoceptor-mediated effects of clonidine.Antazoline shared the ability of phentolamine to stimulate insulin release despite having no or only very little α-adrenoceptor blocking activity. When used under our conditions, it almost entirely failed to alleviate the inhibition of insulin release induced by clonidine.We conclude that the response of the islet cells to imidazoline derivatives is not limited to those capable of blocking α-adrenoceptors. On the other hand, α-adrenoceptor blocking agents of different chemical structure fail to induce the release of additional insulin. We take this as evidence that in our experiments the islet cells respond to imidazoline derivatives and not to α-adrenoceptor blockade.

Phentolamine, a deceptive tool to investigate sympathetic nervous control of insulin release.

SummaryWe investigated the effects of phentolamine and another more selective α2-adrenoceptor antagonist, rauwolscine, on insulin release in vivo (in female Wistar-rats) and in vitro (in perfused rat pancreas and in isolated perifused mouse islets). Phentolamine was found to significantly increase glucose-induced insulin release. On the other hand, rauwolscine failed to do so, when applied in a concentration that effectively antagonized the inhibitory effect of clonidine. These results demonstrate that phentolamine is capable of directly stimulating insulin release. This effect is thus not mediated by α-adrenoceptors. For this reason phentolamine is not an appropriate tool to study possible inhibitory effects of the sympathetic nervous system on insulin release. An enhanced insulin response as may be observed in animals and in man in the presence of phentolamine does not furnish evidence for a tonic inhibitory control of the islet cells by the sympathetic nervous system.

Effects of L-leucine and α-ketoisocaproic acid upon insulin secretion and metabolism of isolated pancreatic islets

LLeucine has been shown to s~uIate insulin release in vivo [ 1,2] and in vitro [3,4] . It is unknown whether Lleucine must be metabolized to elicit this effect. From in vivo experiments Knopf et al. [2] suggested that o-ketoisocaproic acid (o-KHZ) has no hypoglycemic action of its own but must be transaminated to leucine in order to stimulate insulin secretion. Recently the nonmetabolized, but transported, leucine analogue 2-aminobicyclo(2,2,l)heptane-2-carboxylic acid was found to induce insulin release in vivo [5] and in vitro [6] . From these experiments it was concluded that the receptor sites for the stimulation of ins~in release by Lleucine may be transport sites. Since the above-mentioned experiments did not rule out that Lleucine or a-KIC trigger insulin release by metabolic events in the pcell the present studies were performed. (w-KIC induced a marked stimulation of insulin release by isolated pancreatic islets. The accompanying increase of fluorescence of reduced pyridine nucleotides demonstrated prompt changes of islet cell metabolism. In pancreatic islets cr-KIC enhanced the tissue level and the production of leucine. All other tested leucine metabolites did not stimulate insulin release si~~cantly,

Effect of carbohydrates upon fluorescence of reduced pyridine nucleotides from perifused isolated pancreatic islets

SummaryIn perifused pancreatic islets, the fluorescence of reduced pyridine nucleotides (NAD(P)H) was measured continuously. Elevation of glucose concentration in the medium from 0 – 5 mM to 20 mM led to an increase in NAD(P)H-fluorescence beginning 10–20 sec after change of medium. Perifusion with calcium-free media had no influence on this effect. It was, however, partially or completely blocked by 2-deoxy D-glucose, D-glucosamine, or D-mannoheptulose. D-mannose, but not D-fructose and L-lactate, enhanced NAD(P)H-fluorescence from pancreatic islets. Pyruvate caused but a small fluorescence increase. From these observations it is concluded that D-glucose leads to the increase of NAD(P)H-fluorescence by mediation of the phosphoglyceraldehyde dehydrogenase reaction.

Dual action of clonidine on insulin release: suppression, but stimulation when α2-adrenoceptors are blocked

SummaryAs shown previously clonidine reduces glucose-stimulated insulin release and this effect is mediated by inhibitory postsynaptic α2-adrenoceptors.The present experiments demonstrate that clonidine has the additional property to also stimulate insulin release. This became evident when the a2-adrenoceptors of isolated islets were blocked by benextramine, and thus protected from being stimulated by clonidine.In the presence of benextramine, clonidine no longer reduced, but on the contrary enhanced, the release of insulin in response to glucose. In control experiments benextramine by itself failed to affect insulin release from isolated islets.These results show that the imidazoline derivative clonidine shares the property of other imidazoline compounds to enhance the insulin response to glucose. All of these agents may stimulate insulin by binding to “imidazoline-preferring” sites, that clearly differ from α-adrenoceptors.

Studies on the mechanism of l-leucine- and alpha-ketoisocaproic acid- -induced insulin release from perifused isolated pancreatic islets.

SummaryThe insulinotropic effects of L-leucine and α-ketoisocaproic acid have been compared in perifused isolated pancreatic islets. In contrast to α-ketoisocaproic acid (10 mM), L-leucine (10 mM) released less insulin in the presence than in the absence of glucose (5 mM). Changes of islet cell metabolism accompanying insulin release were studied by recording the fluorescence of reduced pyridine nucleotides. The traces of L-leucine-or α-ketoisocaproic acid-induced fluorescence increase differed both in the absence and in the presence of glucose (5 mM). When the medium perif using the islets contained 30 mM L-leucine, α-ketoisocaproic acid (10 mM) still triggered a significant insulin release. These results argue against an indirect action of α-ketoisocaproic acid via transformation to L-leucine. Isocaproic acid (10 mM), L-α-hydroxyisocaproic acid (10 mM) or α-ketoisovaleric acid stimulated no remarkable insulin release, demonstrating that the strong insulinotropic effect of α-ketoisocaproic acid is coupled both to its α-ketogroup and to the length of its carbon chain.

Imidazoline/guanidinium binding sites and their relation to inhibition of KATP channels in pancreatic B-cells

Abstract To elucidate the β-cytotropic effect of imidazoline compounds their inhibitory effect on ATP-dependent K+ channels (KATP channels) in pancreatic B-cells was compared with their binding to membranes from insulin-secreting HIT T15 cells. KATP channels in inside-out patches from B-cells were closed with the following rank order of efficacy at 10 μM: guanabenz > phentolamine = alinidine > clonidine > idazoxan > rilmenidine = amiloride. The last four compounds achieved an incomplete inhibition only. In contrast to sulfonylureas, the inhibitory action of imidazolines was not enhanced by ADP. With intact cells the site which mediates inhibition is less easily accessible for protonated compounds, suggesting a location at the inner face of the plasma membrane. Competition binding experiments were performed by masking α-adrenoceptors and using [3H]clonidine as ligand. Homologous displacement of [3H]clonidine revealed two distinct binding sites in HIT cell membranes characterized by dissociation constants of 38 nM and 4,911 nM and maximal binding capacities of 118 fmol/mg protein and 18 pmol/ mg protein. Generally, ligands for I2 imidazoline receptors were more potent than ligands for I1 imidazoline receptors to displace [3H]clonidine from the high affinity site, which does not fit into the current classification of imidazoline receptors. Binding to the second site had affinities in the micromolar range, similar to the concentrations necessary to inhibit KATP channels in B-cells. However, alinidine and phentolamine inhibited KATP channels already at concentrations at which they displaced [3H] clonidine only from the high affinity site, but not yet from the low affinity site. Since the proportion of the low and high affinity site varied in dependence of the competitor, the imidazoline binding sites in HIT cells may not be independent, but may rather represent two interacting or interconvertible sites both of which may be involved in KATP channel closure.

Thyroxine treatment and insulin secretion in the rat

SummaryThyroxine treatment increases blood glucose and plasma insulin levels in the rat. The hypoglycemic effect of tolbutamide is more pronounced in treated animals. The immediate insulin secretory response of the isolated perfused pancreas to maximal, but not to submaximal, glucose stimuli was increased after thyroxine treatment, especially in the lower dose range. However, as thyroxine treatment reduces insulin release during the prolonged late phase, the total amount of insulin released from the pancreas is reduced. Both the early response to tolbutamide and the subsequent basal secretion were increased after thyroxine treatment. When the pancreas of treated rats was exposed to glucose plus pyruvate the inhibition of the late phase was reversed. Isoprenaline did not overcome the inhibitory effect of thyroxine treatment on the late phase of glucose-induced insulin release. Thyroxine induces a selective inhibition of glucose induced insulin release which is reversed by pyruvate; this indicates that thyroxine interferes with the glycolysis in the beta cell.

A versatile microperifusion system

Abstract A microsystem is described which enables one to correlate secretion from tissue samples with fluorometric recordings of functional parameters and with freezestop measurements of metabolite levels. Small pieces of tissue are placed on nylon gratings or membrane filters and are perifused with different media sequentially while monitoring the tissue fluorescence or collecting the perifusate containing the secretory products. By rapidly pushing a special perifusion chamber into Freon 12 kept at its melting point, the tissue is chilled to −20°C in less than 2 sec. The system has been used to study insulin release and metabolism of isolated pancreatic islets.

Inhibition of insulin and glucagon release from the perfused rat pancreas by cyproheptadine (Periactinol®, Nuran®)

SummaryThe tricyclic compound cyproheptadine (Periactinol®, Nuran®) inhibited glucose-induced insulin release from the perfused rat pancreas. Tolbutamide-stimulated insulin release was significantly reduced in the presence and completely suppressed in the absence of a substimulatory glucose concentration (5 mM). Arginine produced a slow rise of insulin release, which was completely abolished by cyproheptadine. Furthermore the biphasic glucagon release due to the stimulus was inhibited. Oxidation of 14C-glucose in isolated islets was unaltered in the presence of cyproheptadine, and pyruvate added to the perfusion medium failed to reverse the inhibitory effect on glucose induced insulin release, indicating that impaired glucose metabolism is not responsible for the inhibition. In addition, the inhibition remained unchanged when phentolamine was present, suggesting that the effect is not mediated by inhibitory adrenergic alpha receptors. Theophylline, in contrast, partly overcame the inhibition. When the calcium concentration of the medium was enhanced, the inhibitory effect of cyproheptadine was still visible, although the relative inhibition had become smaller. The results suggest that cyproheptadine blocks insulin release by affecting a fundamental step of the stimulus-secretion coupling common to peptide hormones. A participation of a calcium-antagonizing effect in the inhibition is discussed.