Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation.

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Immunoblockade of endogenous glucagon-like peptide-1 by monoclonal antibodies in conscious rats: effect on the insulin response to intragastric glucose.

The physiological action of endogenous active forms of glucagon-like peptide-1 (GLP-1) on the insulin response to intragastric glucose was studied in conscious male Wistar rats by immunoblockade with two monoclonal antibodies directed against different epitopes of GLP-1(7-36)amide. Plasma concentrations of intraperitoneally injected monoclonal antibodies were determined before and during each experiment by an enzyme-linked immunosorbent assay (ELISA) specific for GLP-1-binding antibodies. Three hours after injection of the two monoclonal antibodies, the plasma insulin response (area under the curve) following intragastric glucose 1 g/kg was reduced to a mean level (mean +/- SEM) of 60%+/-8% (n = 11) of control responses previously determined in the same rats, and the time course of the response showed almost no increase in insulin during the first 10 minutes, reaching a maximum of 45.1+/-4.6 microU/mL at 30 minutes, in contrast to the rapid increase of the control response to a maximum of 64.5+/-5.1 microU/mL at 15 minutes. Total C-terminally amidated GLP-1 measured by radioimmunoassay (RIA) of acid ethanol-extracted plasma increased from a mean basal level of 10+/-2 pmol/Lto a peak of 31+/-5 pmol/L at 15 minutes in the control experiments, while basal and response levels greater than 100 pmol/L were recorded after antibody treatment. The increase of plasma glucose was reduced in the presence of the antibodies, peaking at a mean of 9.7+/-0.3 mmol/L at 30 minutes, compared with 11.8+/-0.5 mmol/L at 30 minutes in the control experiments. The action of GLP-1 appears particularly important for the early insulin response to ingested glucose, and the unexpected effect of the antibodies on the glucose response may point to a net promoting effect of GLP-1 on intestinal glucose absorption.

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 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.

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.

Distribution of a novel pituitary protein (7B2) in mammalian gastrointestinal tract and pancreas.

The distribution of a novel pituitary protein (7B2) was determined in the gastrointestinal tract and pancreas of four mammalian species (man, pig, guinea pig, and rat) by a specific radioimmunoassay. The highest concentrations of cross-reacting immunoreactive 7B2 (IR-7B2) were observed in the pancreas and the proximal gut (antrum or duodenum). While the intestinal concentrations varied widely among species, pancreatic IR-7B2 concentrations appeared to be similar in all four species. In the rat, pancreatic islets were found to contain high concentrations of IR-7B2 (5.73 +/- 0.14 fmol/islet, mean +/- SEM). Neonatal capsaicin treatment and enteric nerve section did not affect the concentrations of IR-7B2 in the rat intestine. Layer separation of human gut showed that IR-7B2 is mainly (71 +/- 8%) present in the epithelial fraction. Chromatographic analysis of intestinal and pancreatic extracts from the four species on Sephadex G-100 showed the presence of two immunoreactive peaks at Kav 0.3 and 0.6, but there were both inter- and intraspecies variations in the proportions of the larger and smaller molecular forms.

Acute and short term effects of intestinal alpha-glucosidase inhibition on gut hormone responses in man.

The effect of 50-, 100-, 200-, and 400-mg oral doses of acarbose, a competitive inhibitor of intestinal alpha-glucosidases, on the postprandial release of gut and pancreatic hormones after a 2.2 MJ carbohydrate-rich mixed test meal was determined in five normal subjects according to a double-blind, Latin-square protocol. All the doses of acarbose tested slowed the postprandial plasma glucose rise, without evidence of dose dependency, while maximal inhibition of integrated insulin and gastric inhibitory polypeptide responses to 31 +/- 8% and 28 +/- 7% of control values, respectively, was obtained at the 400-mg dose. The enteroglucagon response was increased to a maximum of 905 +/- 262% of control at the 200-mg dose, and total motilin responses were slightly but not significantly elevated. After one week of regular acarbose administration at 100 mg three times daily, the effects of the 100-mg dose on insulin and enteroglucagon responses were slightly enhanced, and there was no evidence of intestinal adaptation in the form of diminished postprandial endocrine responses. The observed effects are attributed to impairment of carbohydrate digestion in the upper small intestine and suggest that the optimal ratio of desired to unwanted effects is obtained at low doses of acarbose.

Long-term effects of intestinal alpha-glucosidase inhibition on postprandial glucose, pancreatic and gut hormone responses and fasting serum lipids in diabetics on sulphonylureas.

Seventeen non-insulin-dependent diabetics poorly controlled by diet and sulphonylurea drugs took part in a long-term (20-52 weeks) trial of the effect of an alpha-glucosidase inhibitor (acarbose 100 mg thrice daily) on postprandial glycaemic and gastro-entero-pancreatic hormone responses. Patients were assessed before, during, and after the trial period with identical 2.2 MJ mixed test meals plus placebo or acarbose 100 mg, and sulphonylurea therapy was continued throughout. Acarbose administration reduced the integrated postprandial plasma responses of glucose to 58 +/- 10% (mean +/- SEM, p less than 0.001), insulin to 61 +/- 10% (p less than 0.01) and gastric inhibitory polypeptide to 45 +/- 8% (p less than 0.001) of control values, increased the enteroglucagon response to 152 +/- 26% (p less than 0.001) of control and slightly prolonged the postprandial release of motilin. Recorded glycosuria was significantly (p less than 0.01) reduced throughout the treatment period. The effects of acarbose on postprandial glycaemic and endocrine responses remained approximately constant throughout the trial period, and responses returned to pre-treatment values within 2 days of stopping treatment.

Effects of decapeptide of mammalian bombesin and neuromedin B on pancreatic exocrine secretion in the rat.

The effects on pancreatic exocrine secretion of intravenous bolus injections of decapeptide of mammalian bombesin (also called neuromedin C) and neuromedin B, recently isolated mammalian bombesin-like peptides, have been studied and compared with those of amphibian bombesin in anaesthetized rats. Decapeptide of mammalian bombesin and neuromedin B stimulated the volume output from the pancreas with the same potency as that with which they stimulated protein output, as did amphibian bombesin. The maximal peak rates of volume and protein secretion observed in the 5- to 10-min period after the injection of 3 X 10(-10) mol/kg decapeptide of mammalian bombesin were 24.5 +/- 1.2 microliters/5 min and 8.5 +/- 0.5 mg bovine serum albumin equivalents per 5 min (mean +/- SEM, n = 5). These rates were equivalent to those produced by the same dose of amphibian bombesin, but the duration of responses to decapeptide of mammalian bombesin were shorter than those of equimolar doses of amphibian bombesin. The relative potencies of decapeptide of mammalian bombesin and neuromedin B, calculated from the doses producing 50% of maximum effect on total responses, were, respectively, 100 and 0.5% of that of amphibian bombesin. The results suggest that decapeptide of mammalian bombesin, and possibly neuromedin B, could play a regulatory role in the control of exocrine pancreatic secretion.

Glucagon-like peptide-1 does not have a role in hepatic carbohydrate metabolism.

Glucagon-like peptide-1 does not have specific, high-affinity receptors on rat liver membranes, does not displace glucagon from glucagon receptors on these membranes and does not stimulate the production of cyclic AMP by isolated rat hepatocytes. In the presence of glucagon, high concentrations of glucagon-like peptide-1 do not significantly alter the production of cyclic AMP. Thus, glucagon-like peptide-1 appears unlikely to have a direct action on hepatic carbohydrate metabolism.

Molecular forms of glucagon-like peptides in man.

Molecular forms of the glucagon-like peptides (GLP) encoded by the human preproglucagon gene were analysed by chromatography combined with specific radioimmunoassays to the synthetic peptides. Whereas extracts of human pancreas and a glucagonoma contained a large proglucagon cleavage product possessing both GLP-1 and GLP-2 immunoreactivities, extracts of human intestine contained products corresponding to free GLP-1 and a small amount of chromatographically distinct GLP-2 immunoreactivity. It is concluded that post-translational processing of proglucagon differs in pancreas and intestine, so that the C-terminal portion of the molecule is cleaved to liberate free GLP-1 in the intestine. Further processing or degradation results in loss especially of GLP-2 immunoreactivity.

Molecular forms of glucagon-like peptide-1 in human pancreas and glucagonomas.

The structure of human preproglucagon, as deduced from nucleotide sequencing of the preproglucagon gene, contains two glucagon-like peptides (GLP-1 and GLP-2) in the portion C-terminal to glucagon. A rabbit antiserum was raised against synthetic GLP-1-(1-19) which had 20% cross-reactivity with synthetic GLP-1 and des-Gly37-GLP-1 amide, two possible forms of the GLP-1 whole molecule, but no significant cross-reactivity with glucagon or other pancreatic peptides. Immunocytochemistry revealed that the distribution of GLP-1-(1-19) immunoreactivity followed that of glucagon-like immunoreactivity in the normal human pancreas and in two human glucagon-secreting pancreatic tumors. Chromatography of human pancreas extracts on Sephadex G-50 gave peaks of cross-reactivity at Kav values of 0.06-0.16, 0.34-0.39, 0.54-0.58 (the elution position of synthetic GLP-1), and 0.64-0.70. The concentration of immunoreactivity in the Kav 0.54-0.58 peak measured by RIA using GLP-1 or des-Gly37-GLP-1 amide as standard was 94 +/- 7 pmol/g (mean +/- SEM), while the total pancreatic glucagon content was 4.8 +/- 0.8 nmol/g. One extract of a human glucagon-secreting pancreatic tumor contained a prominent peak of GLP-1-(1-19) peptide cross-reactivity with properties identical to those of GLP-1 or des-Gly37-GLP-1 amide on gel filtration and reverse phase high pressure liquid chromatography, but another tumor contained a preponderance of cross-reactive forms of greater molecular size. Pretreatment plasma from three patients with radiological and biochemical evidence of glucagon-secreting tumors contained a peak of cross-reactivity with the chromatographic properties of intact GLP-1. The low concentrations of intact GLP-1 in normal pancreas compared with pancreatic glucagon concentrations suggest that the majority of the proglucagon is cleaved in a manner that does not produce GLP-1, as defined by its delimiting pairs of basic amino acid residues.

Plasma glucagon-immunoreactive components in early life in dogs.

High plasma concentrations of C-terminal immunoreactive glucagon (IRG) have been found during early life in several mammalian species. We have analyzed the plasma IRG of 12 h to 60 day-old dogs in terms of the 4 peaks (IRG greater than 20,000, IRG9000, IRG3500 and IRG2000) obtained by gel filtration on Bio-Gel P-30. Significant changes with age and in response to administered agents were confined to IRG9000 and IRG3500. IRG9000 was 9-fold higher in 12-36 h old dogs than in adults (108 +/- 24 pg/ml pancreatic glucagon equivalents v. 12 +/- 3 pg/ml, mean +/- SEM) and showed a decline to 2-fold higher (27 +/- 5 pg/ml) at 31-60 days. IRG3500 was higher than in the adult only during the first 36 h of life (36 +/- 5 pg/ml v. 15 +/- 3 pg/ml). Arginine infusion (0.5 g/kg over 15 min) caused an increase in plasma levels of both IRG9000 and IRG3500 in the newborn, whereas in adult dogs only IRG3500 was increased. Insulin injection (0.2 U/kg intravenously) causing a marked hypoglycemia had no significant effect on the plasma level of any IRG component in newborn dogs. Dihydrosomatostatin infusion (10 micrograms/kg bolus +/- 90 micrograms/kg over 30 min) caused a decrease in both IRG9000 and IRG3500. The increased basal level and secretory response to arginine of IRG9000 in newborn dogs may reflect an immaturity of the A cells, whereby more of this component, which may represent a precursor of pancreatic glucagon, is secreted than in the adult. The immature A cells also appear to have an impaired secretory response to hypoglycemia.

Pentagastrin-stimulated gastric acid output and plasma enteroglucagon in acarbose-treated rats.

Although the purified porcine enteroglucagons glicentin and oxyntomodulin inhibit pentagastrin-stimulated gastrin acid secretion when given parenterally to rats, it is not known whether the postprandial rise in endogenous enteroglucagons is capable of exerting a similar effect. We have used the alpha-glucosidase inhibitor acarbose in combination with a sucrose- and starch-rich semisynthetic diet over 8 days to bring about a mean increase of 89 pmol/l in the fasting plasma enteroglucagon concentration in rats, without significantly affecting plasma gastrin concentrations. There was no significant suppression of pentagastrin-stimulated gastric acid secretion in the acarbose-treated rats, suggesting that endogenous enteroglucagons do not act as physiological inhibitors of gastric acid secretion.

How glucagon-like is glucagon-like peptide-1?

Although glucagon-like peptide-1 has the appearance of a glucagon-homologue that may be co-secreted with glucagon, synthetic glucagon-like peptide-1-(1-37) does not significantly affect plasma glucose and insulin concentrations when administered at high doses (100 and 400 micrograms) to cortisone-pretreated rabbits. This synthetic preparation thus lacks the primary metabolic effect of glucagon at the doses tested. An intra- or extra-pancreatic role of glucagon-like peptide-1 has yet to be discovered.

The use of a rat isolated ileal preparation to investigate the release of neurotensin.

An isolated and perfused preparation of rat ileum was used to investigate the effects of cholinergic, adrenergic and bombesin stimuli on neurotensin release into the vascular compartment. A vigorous release of neurotensin like immunoreactivity (NTLI) to 200% above basal values in response to intraluminal infusions of emulsified soybean oil (Intralipid) demonstrated the physiological responsiveness of the preparation. Carbachol significantly stimulated the release of both NTLI and bombesin like immunoreactivity (BLI), with maximal responses at 5 X 10(-9) mol/l carbachol of 100% and 400% above basal values for NTLI and BLI, respectively. Noradrenaline at 10(-6) and 10(-4) mol/l caused no significant release of NTLI but markedly inhibited spontaneous BLI release. Synthetic amphibian bombesin caused a marked release of NTLI to 81% and 100% above basal at infusion concentrations of 5 X 10(-11) and 5 X 10(-10) mol/l respectively. These results suggest that there is either a direct effect of cholinergic agents on the N cells bringing about NTLI release, or an indirect effect via the release of bombesin like peptides from intrinsic gut neurones. This preparation provides a useful model to study the complex neural, paracrine and endocrine interactions in the gastrointestinal tract.

The stimulus-secretion coupling of glucose-induced insulin release. LIII. Calcium-dependency of the cyclic AMP response to nutrient secretagogues.

Above a threshold value in excess of 5.6 mM, D-glucose increases the amount of cyclic AMP measured by radioimmunoassay in pancreatic rat islets and their surrounding incubation medium. As judged from the cyclic AMP content of islets exposed to isobutylmethylxanthine (1.0 mM), the glucose-induced increment in the rate of cyclic AMP generation represents a rapid and sustained phenomenon. The stimulant action of glucose on cyclic AMP accumulation is mimicked by L-leucine, and L-glutamine, these amino acids acting synergistically of one another. Trifluoperazine slightly decreases but fails to abolish the effect of glucose. In the absence of extracellular Ca2+, however, the cyclic AMP response to D-glucose, L-leucine and/or L-glutamine is severely impaired. These findings are compatible with the view that an increase in the generation rate of cyclic AMP participates in the process of nutrient-stimulated insulin release. This increase could be secondary to the nutrient-induced accumulation of Ca2+ in the islet cells leading to activation of adenylate cyclase by calmodulin.

PHI stimulates intestinal fluid secretion.

Regulatory peptides are likely to have a role in the control of net intestinal fluid transport. PHI is a peptide recently isolated from porcine duodenum which has been shown to have sequence homologies with other peptides of the glucagon-secretin family. We have studied the effect of intravenous infusion of synthetic PHI on net intestinal fluid transport in the rat small intestine. During PHI infection net absorption was reduced in the duodenum and jejunum and net secretion was observed in the ileum. Thus synthetic PHI appears to be capable of strongly stimulating intestinal secretion in the rat.

Renal metabolism of gut glucagon-like immunoreactivity.

To examine the role of the kidney in the catabolism of gut glucagon-like immunoreactivity (GLI), we compared the plasma GLI responses of normal and nephrectomized dogs given intraduodenal glucose loads and studied the clearance of gut GLI by the isolated perfused rat kidney. Both basal and postload plasma samples were analyzed with a glucagon C-terminal specific antibody and a GLI-reacting N-terminal antibody. The GLI response was taken to be the difference between the increments seen with these two antibodies after glucose loading. Although glucose-induced GLI increments could not be detected in untreated plasma from nephrectomized dogs, chromatographed plasma revealed a significant rise in GLI-fraction II (7000--12000 daltons) in both nephrectomized and normal dogs (732 +/- 200 and 586 +/- 111 pg/ml, respectively). We also found that crystalline glucagon was cleared by the isolated closed-circuit perfused rat kidney, but gut-GLI either as crude extract or as peak I (7000--12000 daltons) was not. Our data suggest that the kidney may not play an important role in gut-GLI catabolism.