Role of Ca2+ and protein kinase C in the receptor-mediated activation of Na+/H+ exchange in isolated liver cells.

This work aimed to study the relationship between agonist-induced changes in cytosolic free calcium levels, protein kinase C (PKC) activity and intracellular pH in isolated liver cells. We observed that, like alpha1-adrenergic agonists, the Ca2+-mobilizing vasoactive peptides vasopressin and angiotensin II produced an extracellular-Na+-dependent, 5-(N-ethyl-N-isopropyl)amiloride-sensitive, intracellular alkalinization, indicative of Na+/H+ antiporter activation. Blocking the agonist-induced increase in the intracellular Ca2+ concentration using the calcium chelator bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid (BAPTA) prevented all types of receptor-mediated intracellular alkalinization. Thus activation of the Na+/H+ exchanger by either alpha1-adrenergic agonists or vasoactive peptides relies on the mobilization of intracellular Ca2+. In contrast, only the alpha1-adrenergic-agonist-induced alkalinization was dependent on extracellular Ca2+. Even though alpha1-adrenergic as well as vasoactive peptide agonists stimulated protein kinase C (PKC) activity in isolated liver cells, only the alpha1-adrenoreceptor-mediated intracellular alkalinization was dependent on PKC. According to these observations, Ca2+-mobilizing agonists appear to activate the Na+/H+ exchanger by at least two different mechanisms: (1) the alpha1-adrenoreceptor-mediated activation that is dependent on extracellular Ca2+ and PKC; and (2) vasoactive-peptide-induced alkalinization that is independent of extracellular Ca2+ and PKC. The alpha1-adrenoreceptor-mediated, PKC-sensitive, activation of the Na+/H+ exchanger seems to be responsible for the distinct ability of these receptors to elicit the sustained stimulation of hepatic functions.

Identification of amidated forms of GLP-1 in rat tissues using a highly sensitive radioimmunoassay.

The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7-36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7-36)amide and GLP-1(1-36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7-36)amide, GLP-1(1-36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7-36)amide could be identified in the hypothalamus, in amounts that represented 55-94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1-36)amide, representing 0.8-3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7-36)amide (0-0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7-36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27-73% of total GLI, and in five of six specimens only GLP-1(7-36)amide could be identified on HPLC, in amounts representing 13-17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1-36)amide (13% of total GLI) in addition to GLP-1(7-36)amide (31% of total GLI). Acid-ethanol extraction of peptide-free rat plasma with added GLP-1(7-36)amide gave recoveries of 91+/-SEM 2% in the range 20-200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1+/-1.1 pmol/l and rose to a maximum of 15.4+/-3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.

Effect of (8-32) salmon calcitonin, an amylin antagonist, on insulin, glucagon and somatostatin release: study in the perfused pancreas of the rat.

1. The 8-32 fragment of salmon calcitonin ((8-32) sCT) has been proposed as a highly selective amylin receptor antagonist. 2. In the present study, we have studied the influence of (8-32) sCT on the inhibitory effect of both amylin and its structural congener, calcitonin gene-related peptide (CGRP), on insulin secretion in the rat perfused pancreas. 3. Both amylin and CGRP, at 75 pM, clearly inhibited glucose-induced insulin release (by 80% and by 70%, respectively). Simultaneous infusion of 10 microM (8-32) sCT reversed the inhibitory effect of amylin (by 80%; P < 0.05 vs. amylin experiments) but did not significantly affect the inhibition of glucose-induced insulin output elicited by CGRP. Furthermore, at the same concentration (10 microM), (8-32) sCT alone potentiated the insulin response to 7 mM glucose (2.5 fold; P < 0.05) whilst it did not affect glucagon or somatostatin secretion. 4. The observation that infusion of an amylin antagonist into the rat pancreas potentiates the insulin response to glucose, favours the concept of endogenous amylin as an inhibitor of insulin release. 5. Finally, as an amylin antagonist at the level of the beta-cell, (8-32) sCT might be considered of potential interest in experimental and clinical pharmacology.

Inhibitory effect of amylin (islet amyloid polypeptide) on insulin response to non-glucose stimuli. Study in perfused rat pancreas.

Amylin, also called islet amyloid polypeptide (IAPP), can inhibit the glucose-induced insulin secretion in perfused rat pancreas at 75 pmol/l, a concentration comparable to that found in the effluent of this experimental model. To further explore the influence of amylin on insulin release, we investigated the effect of synthetic rat amylin (75 pmol/l) on insulin response to non-glucose secretagogues. These agents stimulate B-cell secretion via different mechanisms, such as a dihydropyridine derivative (BAY K 8644, 10 mmol/l) which activates Ca(2+)-channels, a sulfonylurea (tolbutamide, 0.2 mmol/l) which blocks ATP-dependent K(+)-channels, KCL (11 mmol/l) which depolarizes B cells and the 26-33 fragment of cholecystokinin (8-CCK, 1 nmol/l) which increases phospholipid turnover. The study was performed in perfused rat pancreas. Amylin significantly inhibited insulin response to BAY K 8644 (65%), KCI (60%) and 8-CCK (80%) as well as the early phase of tolbutamide-induced insulin output (70%). Thus, amylin can inhibit insulin release induced by secretagogues that interact at different levels of B-cell stimulus-secretion coupling. This inhibition may be due to a multifarious influence of amylin on the B-cell secretory mechanism and/or a disturbing effect on a distal, crucial step in the insulin-releasing mechanism, e.g. by affecting exocytosis of the secretory granule or by inhibiting an essential metabolic pathway within the B cell.

Effect of lithium on plasma glucose, insulin and glucagon in normal and streptozotocin-diabetic rats: role of glucagon in the hyperglycaemic response.

1. Lithium salts, used in the treatment of affective disorders, may have adverse effects on glucose tolerance in man, and suppress glucose-stimulated insulin secretion in rats. 2. To study the interaction of these effects with pre-existing diabetes mellitus, plasma glucose and insulin responses to lithium chloride were measured in male Wistar rats made diabetic with intraperitoneal streptozotocin, and in normal controls. 3. In both normal and diabetic anaesthetized rats, intravenous lithium (4 mEq kg-1) caused a rise in plasma glucose. In absolute terms, the rise was greater in diabetic (5.2 mmol l-1) than in normal rats (2.3 mmol l-1). 4. Plasma insulin concentrations were reduced by lithium in normal rats, but the low insulin concentrations measured in the diabetic rats were not significantly changed. 5. After intravenous glucose (0.5 g kg-1), lithium-treated diabetic rats showed a second rise in plasma glucose at 60-90 min without any insulin response, while normal rats showed typically reduced insulin responses and initial glucose disappearance rates. 6. Intravenous glucose reduced plasma glucagon concentrations to a greater extent in normal than in diabetic rats, but lithium induced an equal rise in plasma glucagon in both groups, with a time-course similar to that of the hyperglycaemic effect. 7. The hyperglycaemic action of lithium is greater in the hypoinsulinaemic diabetic rats and appears to involve a stimulation of glucagon secretion in both normal and diabetic animals.

Amylin (islet amyloid polypeptide) inhibition of insulin release in the perfused rat pancreas: implication of the adenylate cyclase/cAMP system.

Amylin inhibits glucose-induced insulin secretion in the rat pancreas. To study the mechanism by which amylin acts on the B-cell, we have investigated, in the perfused rat pancreas, the effect of synthetic rat amylin (75 pM) on insulin release elicited by secretagogues acting on the B-cell via the adenylate cyclase/cAMP system, i.e., glucagon (10 nM), gastric inhibitory polypeptide (GIP, 1 nM), forskolin (1 microM) and isobutylmethylxanthine (IBMX, 75 microM). In addition, we examined the effect of amylin on GIP-induced insulin release in pancreata from rats pretreated with pertussis toxin, an agent which inactivates certain Gi proteins coupled to adenylate cyclase. Amylin inhibited the insulin response to glucagon (approx. 70%), GIP (approx. 90%), IBMX (approx. 75%) as well as the early phase of forskolin-induced insulin output (approx. 74%). However, amylin failed to modify GIP-induced insulin release in pancreata obtained from pertussis toxin pretreated rats. These results would indicate that the inhibitory effect of amylin on insulin secretion could be, at least in part, attributed to its interfering with the adenylate cyclase/cAMP system. Furthermore, prevention of the inhibitory effect of amylin on GIP-induced insulin output by pertussis toxin pretreatment, supports the concept that amylin can inhibit insulin release via a pertussis toxin-sensitive Gi protein coupled to the adenylate cyclase system.

Effect of pertussis pretreatment on plasma glucose and insulin responses to lithium in rats.

1. Administration of lithium to rats causes a rise in plasma glucose and suppresses glucose-stimulated insulin secretion. These effects are blocked by the alpha 2-adrenoceptor antagonist, yohimbine. 2. Pretreatment of rats with Bordetella pertussis toxin resulted in a reversal of the usual plasma glucose and insulin responses to intravenously administered lithium (4 mEq kg-1). There was a slow fall in plasma glucose, while plasma insulin rose to 267 +/- 42% (+/- s.e.mean) of control values at 30 min. The effect of lithium on glucose-stimulated insulin secretion was also reversed; there was a marked increase in the insulin response which contrasted with the suppression seen in normal controls. 3. In perifused islets of Langerhans isolated from pertussis pretreated rats, the previously described inhibition by lithium of the second phase of glucose-stimulated insulin secretion from normal islets was almost completely abolished. 4. The results are consistent with the hypothesis that these effects of lithium are mediated by the influence of catecholamines on the islets. When the inhibitory effect of alpha 2-adrenoceptors is abolished by pertussis treatment, which blocks the action of the inhibitory guanine nucleotide-binding protein Gi, effects of beta-adrenoceptor stimulation predominate, leading to an increased secretion of insulin.

Role of adrenoceptors in vitro and in vivo in the effects of lithium on blood glucose levels and insulin secretion in the rat.

1. Pretreatment of rats with the non-selective alpha-adrenoceptor antagonist dihydroergotamine counteracts the inhibition of glucose-induced insulin secretion caused by lithium both in vitro and in vivo. The present study was therefore carried out to specify further which type of adrenoceptor is involved in lithium-induced hyperglycaemia and inhibition of insulin secretion. 2. The lithium-induced effects were reversibly blocked by pretreatment of rats with the alpha 2-adrenoceptor antagonist yohimbine or a combination of yohimbine and the non-selective beta-receptor antagonist propranolol, whereas the alpha 1-receptor antagonist prazosin and propranolol alone were ineffective in blocking these effects. 3. These findings suggest that the effects of lithium on plasma glucose and insulin levels are mediated mainly by the stimulation of alpha 2-adrenoceptors.

Dihydroergotamine, but not naloxone, counteracts lithium as an inhibitor of glucose-induced insulin release in isolated rat islets in vitro.

Lithium exerts an inhibitory effect on glucose-induced insulin release. Lithium (5 mmol/l) added 30 min prior to glucose stimulation or together with glucose (16.7 mmol/l) failed to affect first phase, but reduced second phase glucose-induced insulin release by 35%. Similar results were obtained when islets isolated from rats following long-term oral lithium treatment were perifused with glucose (16.7 mmol/l). The inhibitory effect of lithium was counteracted by pretreatment of the rats with the alpha-adrenergic blocking agent dihydroergotamine, whereas the opiate antagonist naloxone had no apparent effect on lithium-induced inhibition of glucose-stimulated insulin release.

Blocking effect of naloxone, dihydroergotamine and adrenalectomy in lithium-induced hyperglycaemia and glucose intolerance in the rat.

The direct effect of lithium administration on plasma glucose levels and glucose-induced insulin release, and the role of opioid and amine systems in these effects were examined in rats. Naloxone, an opiate antagonist, and dihydroergotamine, an alpha-adrenergic blocking agent, reversed the hyperglycaemia as well as the inhibition of glucose-stimulated insulin release induced by lithium. In adrenalectomized rats, administration of lithium induced hypoglycaemia and not hyperglycaemia as in the intact rats. The results suggest that the interaction of secreted endorphins with the sympathetic nervous system is the likely cause of the hyperglycaemia and the inhibition of the glucose-stimulated insulin release induced by lithium.

Incorporation of (U-14C)-glucose into glycogen in normal rat pancreatic islets.

The incorporation of glucose into glycogen was determined in pancreatic islets isolated from normal rats and incubated with glucose (5 or 20 mM) and compounds known to affect glycogen metabolism in other tissues. Incubation of pancreatic islets with glucose (20 mM) induced a marked increase in radioactive glycogen. Exposure to epinephrine in the presence of glucose (20 mM) slightly increased incorporation of glucose into glycogen. In contrast the incorporation of glucose into glycogen was not affected when isolated islets were exposed to glucagon or insulin, whereas anti-insulin serum in the incubation medium decreased radioactive glycogen formation.

"Big big" gastrin release by the isolated islets of Langerhans incubated in vitro.

The immunoreactive form of gastrin released by the islets and some of the characteristics of this release have been studied. This gastrin released by the islets in the present experiments corresponds to what has been named "Big Big" gastrin in serum of patients with the Zollinger-Ellison syndrome, in normal human serum and in extracts of proximal jejunum. Most of the "Big Big" gastrin released from the islets corresponds to spontaneous release.