Article

Effect of epinephrine and mannoheptulose on early and late phases of glucose-stimulated insulin release

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Previous studies have revealed the existence of two separate phases in the glucose-stimulated insulin release. Exposure of microdissected mouse islets to mannoheptulose or epinephrine effectively inhibited both the initial transient release and the second persistent phase of insulin secretion. These results suggest that metabolism of glucose is a prerequisite for both phases of insulin release. The current view of mannoheptulose as an inhibitor of the β-cell phosphorylation of glucose was corroborated by the observation that this drug prevented the increase of fructose-1,6-diphosphate normally observed with increasing glucose levels.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... That transition from one steady-state to another was more sluggish in intact islets than in dispersed islet cells is also evident from the experiments with epinephrine. Epinephrine is a known inhibitor of glucose-stimulated insulin release [1,9] and it suppressed secretion from the dispersed cells as well as from the intact islets. However, the return to the basal secretory rate occurred faster in the dispersed cells than in the intact islets. ...
Article
The overall dynamics of glucose-induced insulin release was strikingly similar in dispersed cells and intact islets perifused in parallel. Both preparations exhibited a latency of 1-2 min, after which period there was a brisk rise of insulin release followed by a sustained second phase. During the second phase, insulin release from dispersed cells attained a stable plateau rate, whereas the release from intact islets continued to rise. Epinephrine (1 muM) inhibited the release in both preparations, but the return to basal rate was faster in the dispersed cells than in the intact islets. The dispersed cells oxidized glucose at a constant rate for at least 60 min; the glucose oxidation was markedly sensitive to changes of the glucose concentration in the range of 3-20 mM.
Article
d-Glyceraldehyde stimulated the release of insulin from pancreatic islets of Umeå- ob ob-mice whether or not glucose was present in the medium. Like the action of glucose, that of d-glyceraldehyde was biphasic in time, exhibited a sigmoidal dose-response relationship, was potentiated by theophylline, arginine, iodoacetamide, or l-glyceraldehyde, and was inhibited by epinephrine, 2,4-dinitrophenol, or Ca2+ deficiency. Half-maximum and maximum stimulations were produced by about 3 mm and 10 mm d-glyceraldehyde. Positive interactions were observed between 5 mm d-glyceraldehyde and 5 mm glucose and between 10 mm d-glyceraldehyde and 10 mm leucine. Mannoheptulose (10 mm) or glucosamine (10 mm) did not inhibit but potentiated the effect of 10 mm d-glyceraldehyde. Dihydroxyacetone (2.5-20 mm) also initiated insulin release in the absence of glucose. On the other hand, 5-10 mm l-glyceraldehyde did not initiate secretion but potentiated the effects of 5 mm glucose or 5 mm d-glyceraldehyde. d-Glyceraldehyde or dihydroxyacetone reduced the production of 14CO2 from d-[U-14C]glucose; l-glyceraldehyde had a smaller and statistically insignificant effect. The results suggest that by being phosphorylated and entering glycolysis in the β-cells, d-glyceraldehyde and dihydroxyacetone act as functional analogues of glucose as secretory stimulus. Initiation of insulin release by glucose, d-glyceraldehyde, or dihydroxyacetone may thus depend on the production of a metabolic signal at or below the triose phosphate level.
Article
Isolated rat islets were maintained in vitro in a simple perifusion system and the rate of insulin secretion was maintained throughout the period of perifusion. Exposure of the perifused islets to alloxan (20 mg. %) for a period of 5 min produced complete inhibition of subsequent glucose induced insulin secretion with retention of basal secretion. Glucose, mannose and 3 0 methyl glucose provided complete or almost complete protection of the perifused islets from the inhibitory effect of alloxan on glucose induced insulin release. The order of potencies of various hexoses to protect against alloxan toxicity in vitro was glucose (100 ≥ 3-0 methyl glucose ≥ mannose > 2 deoxyglucose > galactose > fructose >> L glucose (0). Mannoheptulose diminished but did not abolish the protective effect of glucose against alloxan. The first phase of tolbutamide induced secretion was retained after exposure to alloxan. This finding indicated that the beta cells were still viable and suggests that a separate receptor or transport site for tolbutamide may exist on the beta cell. The possible mechanisms by which the hexoses protect the beta cells from alloxan are discussed.
Article
The transport and oxidation of glucose, the content of fructose 1,6 diphosphate, and the release of insulin were studied in microdissected pancreatic islets of ob/ob mice incubated in Krebs Ringer bicarbonate medium. Under control conditions glucose oxidation and insulin release showed a similar dependence on glucose concentration with the steepest slope in the range 5-12 transport. However, Ca 2+ or Na + deficiency glucose was inhibited. The inhibitory effects of Na + deficiency on the secretory responses to different concentrations of glucose correlated with those on 14CO 2 production. When islets were incubated with 17 mM glucose, the sudden replacement of Na + by choline ions resulted in a marked but transient stimulation of insulin release that was not accompanied by a demonstrable increase of glucose oxidation. Galactose and 3 O methylglucose had no effect on glucose oxidation or on insulin release. The results are consistent with a metabolic model of the β cell recognition of glucose as insulin secretagogue and with the assumption that Ca 2+ or Na + deficiency, or the addition of adrenaline or diazoxide, inhibit insulin release at some step distal to stimulus recognition. In addition the results suggest that these conditions create a partial metabolic block of glycolysis in the β cells. Hence the interrelationship between the processes of stimulus recognition and insulin discharge may involve a positive feedback of secretion on glucose metabolism.
Article
Full-text available
The effects of p-chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid on pancreatic islets were studied in vitro. Obese-hyperglycaemic mice were used as the source of microdissected islets containing more than 90% beta-cells. p-Chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid stimulated insulin release at concentrations of 0.01mm or above. This stimulation was significantly inhibited by the omission of Ca(2+) or the addition of adrenaline, diazoxide or 2,4-dinitrophenol. p-Chloromercuribenzoic acid or chloromercuribenzene-p-sulphonic acid did not interfere with the insulin-releasing ability of glucose. Micro-perifusion experiments revealed that the release of insulin in response to organic mercurial occurred almost instantaneously, was reversible, and was biphasic. The two mercurials inhibited glucose transport as well as glucose oxidation, and increased the mannitol and sucrose spaces of isolated islets. Compared with the effects on insulin release, those on glucose transport and membrane permeability were characterized by a longer latency and/or required higher concentrations of organic mercurial. Apart from a seemingly higher proportion of beta-cells exhibiting certain degenerative features, in islets exposed to 0.1mm-chloromercuribenzene-p-sulphonic acid for 60min, no significant differences with respect to beta-cell fine structure were noted between non-incubated islets and islets incubated with chloromercuribenzene-p-sulphonic acid or glucose or both. It is suggested that insulin release may be regulated by relatively superficial thiol groups in the beta-cell plasma membrane.
Article
beta-Cell-rich pancreatic islets were microdissected from ob/ob-mice and loaded with 45Ca in the presence of 3 or 20 mM glucose. Subsequent measurements of the effluxes of radioactivity in a perifusion apparatus revealed that the slowly exchangeable 45Ca taken up in response to glucose was also preferentially mobilised by this compound. Glucose stimulation of 45Ca efflux was abolished after omission of calcium from the perifusion medium but persisted when insulin release was inhibited by prolonged starvation, addition of L-epinephrine or lowering of temperature. The presence of a stimulated efflux of radioactivity even under conditions of inhibited insulin release indicates that sources other than beta-granules ejected by exocytosis contribute to the additional 45Ca released after raising the glucose concentration of the perifusion medium. It is suggested that the beta-cell depolarisation as such may account for part of the 45Ca mobilised by glucose.
Article
At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose concentrations, iodoacetamide exerted both an initial stimulatory and a subsequent inhibitory action. When islets were perifused with 1mm-iodoacetamide and 17mm-glucose the inhibitory action predominated after about 15min of transient stimulation. With decreasing concentrations of iodoacetamide the stimulatory phase was gradually prolonged, and with 0.003-0.1mm-iodoacetamide stimulation only was observed for 75min. Prolonged stimulation was also noted after a short pulse of iodoacetamide. Similar responses to 0.1mm-iodoacetamide were observed with islets from normal mice. With islets from ob/ob-mice the effect of 0.1mm-iodoacetamide was reproduced with 0.1mm-iodoacetate, whereas 0.1mm-acetamide had no apparent effect. Iodoacetamide increased the V(max.) of glucose-stimulated insulin release without altering the apparent K(m) for glucose. Leucine, glibenclamide or theophylline could not replace glucose in this synergistic action with iodoacetamide. Iodoacetamide rather inhibited the insulin-releasing action of theophylline. Iodoacetamide-induced potentiation of the glucose-stimulated insulin release was rapidly and reversibly inhibited by mannoheptulose, adrenaline, or calcium deficiency. The potentiating effect on insulin release was not paralleled by effects on glucose oxidation or on islet fructose 1,6-diphosphate. However, the inhibitory action of iodoacetamide might be explained by inhibition of glycolysis as evidenced by an inhibition of glucose oxidation and a rise of fructose 1,6-diphosphate. The results support our previous hypothesis that thiol reagents can stimulate insulin release by acting on relatively superficial thiol groups in the beta-cell plasma membrane. Glycolysis seems to be necessary in order for iodoacetamide to stimulate in this way.
Article
In den Ländern, in denen eine hochkalorische Ernährung die Regel ist, nehmen Adipositas und Diabetes mellitus immer mehr an Häufigkeit zu. Obgleich in den vergangenen Jahren eine Fülle von Informationen über die ursächlichen Faktoren und die Verlaufsformen dieser Stoffwechselstörungen erbracht wurden, blieben viele Fragen offen, deren Beantwortung aus technischen oder ethischen Gründen am Menschen schwierig oder unmöglich ist. Erst die Züchtung genetisch fettsüchtigerund/oder hyperglykämischer Laboratoriumstiere erbrachte Modelle, die es gestatten, Stoffwechselbesonderheiten des zur Fettsucht oder zur Zuckerkrankheit neigenden Organismus intensiv zu studieren, um so mögliche Rückschlüsse auf diese Stoffwechselstörungen beim Menschen zu ziehen.
Article
An in vitro system with microdissected mouse islets was employed for studying insulin release. Islets from obese-hyperglycemic mice were considered particularly useful in view of their high content of adequately functioning β-cells. After freeze-drying and weighing each of the incubated islets it was possible to express the rate of insulin release per islet dry weight. Insulin released from a single incubated islet could be measured radioimmunologically using crystalline mouse insulin as standard. The effects of various factors were examined, including diffusion of released insulin, islet size, shaking, temperature, pH, osmotic strength, oxygen tension, and treatment with enzymes. Provided that the basal rate was kept low, reproducible values could be obtained for both the early and the late phases of the glucose-stimulated insulin release. Exposure of the microdissected islets to collagenase tended to increase the basal rate of insulin release but did not significantly affect the total insulin response to glucose nor the concomitant rise in the islet level of fructose-1,6-diphosphate. The present in vitro system offers a useful model for pharmacological screening on the insulin-releasing capacity of various compounds. It is also important that the rate of insulin release from the individual islet can be directly related to its metabolism.
Article
A dose-response study of the inhibitory effect of epinephrine on glucose-induced insulin secretion in man has been performed. The results demonstrate the exquisite sensitivity of the pancreatic β-cells to epinephrine, as small a dose as 3 mμg/kg/min inducing a 50% inhibition. These studies emphasize the physiological significance of epinephrine for the regulation of insulin secretion. They also show that in previous studies in man and animals supramaximal amounts of epinephrine have been employed. Our results also suggest that the actions of epinephrine and glucose on insulin secretion are competitive. Infusion of aminophylline had only a minor inhibitory effect on the action of epinephrine. Therefore, our data do not permit the conclusion that epinephrine suppresses insulin release exclusively by diminishing the rate of synthesis of cyclic AMP. Other possible mechanisms are discussed.
Chapter
Es würde zu weit führen, die sehr umstrittenen Fragen nach der Spezifität biologischer oder immunologischer Insulin-Bestimmungsverfahren sowie laboratoriums- und anwendungsbezogene Vorteile und Nachteile einzelner Methoden selbst hier im einzelnen zu diskutieren. Immunchemisch wird im Serum sicher nur Insulin und, abhängig von der Spezifität der verwendeten Antiseren, zugleich auch Proinsulin gemessen. Die üblicherweise durch Immunisierung von Meerschweinchen gewonnenen Antiinsulinseren erfassen außer Proinsulin sehr wahrscheinlich auch — soweit diese im Serum oder Plasma vorkommen — weitere Insulinvorstufen, wie Desdipeptid-Proinsulin und die Arginin-Insuline. Außer immunchemisch reagierendem Insulin (IRI) bzw. Insulin-ähnlichem immunoreaktivem Material kann in Serum oder Plasma biologisch Insulin-ähnliche Aktivität (ILA) nachgewiesen werden.
Article
The impact of the catecholamine epinephrine and the postulated inhibitory second messenger prostaglandin E(2) (PGE(2)) on the kinetics and magnitude of glucose-induced insulin secretion were compared and contrasted. In agreement with a number of studies, epinephrine was a most effective antagonist of glucose-induced insulin secretion. Dose-response studies using 8 to 10 mmol/L glucose as stimulant established that levels as low as 1 to 10 nmol/L of the catecholamine were effective at inhibiting release. Glucose (20 mmol/L) caused an approximately 25-fold increase in insulin secretion, an effect that was completely abolished by 1 micromol/L epinephrine. Under conditions where it completely abolished 20 mmol/L glucose-induced insulin release, epinephrine (1 micromol/L) reduced, but did not abolish, the stimulatory effect of glucose on phospholipase C activation. Chronic 3-hour exposure to 10 mmol/L glucose alone desensitized the islet to subsequent stimulation by glucose. Despite its ability to completely suppress secretion to 10 mmol/L glucose, epinephrine failed to protect the islet from hyperglycemia-induced desensitization. In sharp contrast to epinephrine, PGE(2) at levels ranging from 1 to 10 micromol/L had no discernible adverse effect on 10 mmol/L glucose-induced secretion. These findings suggest that multiple mechanisms contribute to the inhibitory impact of epinephrine on release and, in conjunction with other studies, cast serious doubt on the concept that PGE(2) plays any significant inhibitory role in the regulation of glucose-induced secretion.
Article
A technique for the dissection of fresh pancreatic islets of different mammalian species is described. Besides being suitable for microchemical analyses, the isolated islets seem of potential value for direct studies of the metabolism of the islet cells. The method has proved to be useful for the isolation of relatively large amounts of islet tissue for analytical purposes.
Article
Summary With a total volume of only a few per cent of the whole gland the mammalian endocrine pancreas is dispersed in a great number of islets of Langerhans. Studies of this endocrine organ are further complicated by the fact that each islet is composed of cells representing different endocrine functions. The present communication deals with some attempts to overcome these analytical difficulties by the following experimental approaches: A) The observation of a strict balance between the number of large and small islets made it possible to introduce rapid methods for estimation of the total islet volume. It was found necessary merely to count the number of large islets in pancreatic sections taken at regular intervals. There was also a linear relationship between the total islet volume and the number of long islet intercepts obtained by scanning sections with parallel lines. B) Attempts were made to identify the different types of islet cells by restaining thin paraffin sections after initial silver impregnation. Most important for the final success was the elaboration of a technique which gave a consistent argyrophilia in some islet cells, and the observation that these silver deposits could be removed by oxidation in potassium permanganate. It is now well established that the pancreatic islets contain 1, 2 and -cells as well as some cells which lack discernible granules both in the light and electron microscope. C) Evaluation of how various substances affect insulin release was simplified by the introduction of a system in vitro employing a single islet microdissected from an obese-hyperglycaemic mouse. This technique allowed both a description of insulin release in terms of islet weight, and a correlation of the rate of insulin secretion with other metabolic events in the -cells. The amount of insulin released during 30 min was found to be about one per thousand of the islet dry weight, or less than one per cent of the -cell content of insulin. The rate of insulin secretion was significantly enhanced by increasing the movements of the incubation medium by shaking. A water extract of the 1-cells served as an effective inhibitor of insulin release. D) A combined approach in vivo and in vitro was found to provide a useful system for analyzing how the -cell levels of glycolytic intermediates and cofactors were related to the rate of insulin secretion. After the freeze-dried pancreas sections had been exposed to formaldehyde vapours and refixed in Bouin's solution, the islet cells could be identified by silver impregnation and restaining. The sulphonylurea-stimulation of insulin release was found to be associated with a significant depression of the -cell content of ATP and glycogen. There was, on the other hand, a striking accumulation of fructose-1.6-diphosphate and other intermediates of glucose above this metabolic stage when insulin secretion was inhibited either by epinephrine or diazoxide or by omission of Ca2+. These data were tentatively interpreted as indicating the existence of a rate limiting step in -cell glycolysis of direct significance for regulation of insulin release. This control site might be the sequence phosphoglyceraldehyde dehydrogenase-phosphoglycerate kinase.
Article
Quantitative histochemical measurements of glycolytic intermediates and cofactors in samples of the order of 0.1 µg dry weight have been made possible by the use of enzymatic fluorometric methods in combination with the technique of enzymatic cycling. In order to prevent evaporation of the necessarily small volumes of fluid and to avoid exposure to CO2 in the air, incubations are carried out under oil in wells drilled in Teflon. The general technique for conducting analyses in oil wells is described together with specific procedures for measuring glycogen, glucose, glucose 6-phosphate, uridine diphosphoglucose, fructose diphosphate, lactate, adenosine triphosphate, phosphocreatine and inorganic phosphate. The potential of the technique is illustrated by measurements of these compounds in each of nine discrete layers of the retina.
Article
Insulin secretion induced in vitro by glucose in pancreatic tissue of the rat is unaffected by addition of phloridzin and 3-0-methylglucose to the incubation media, suggesting that the beta-cells are freely permeable to glucose. D-glucosamine and mannoheptulose markedly inhibit the stimulant effect of glucose upon insulin secretion. The inhibitory effect of mannoheptulose is reversible and of the competitive type. Mannoheptulose also competitively inhibits the phosphorylation of glucose by isolated islet homogenates. Phosphorylation of glucose thus appears as a necessary step in the stimulation of insulin secretion by this sugar.
Article
The dynamics of insulin release in response to relatively long infusions of glucose were studied in the isolated perfused rat pancreas. Insulin secretion was determined by immunochemical assay of the total portal vein effluent. Histological examination of the perfused pancreases and measurement of oxygen consumption by these tissues indicated that optimal physiological conditions were used. It was observed that, when glucose was infused for a period of approximately 1 hr into a perfused pancreas, there appeared 2 distinctly different phases of insulin release. There was an early, or rapid, release of insulin which subsided within approximately 2 min, followed by a late, or slow release phase which continually increased in rate until termination of the glucose infusion. The contribution of newly synthesized insulin to either phase was determined by comparing the insulin release by normal control preparations to that by preparations which were treated with puromycin. Incorporation of L-valine-14C was used a...
Article
The pancreatic islets in obese-hyperglycemic mice display a normal response of insulin secretion when stimulated with glucose . This indicates that the impaired glucose metabolism is due to extra-pancreatic factors rather than to deficient β-cell function. In fact, the pancreas of these mice is a useful source from which to isolate large numbers of mammalian β-cells suitable for studies of the synthesis and secretion of insulin. The very large islets secreted less insulin than the normally sized ones. Whether this can be attributed to a low concentration gradient for insulin in the central part of the large islets or to the presence of α1-cells remains obscure.
Article
Plasma insulin concentration was measured during a standardized glucose infusion test (GIT) in 85 healthy subjects with a normal glucose tolerance and in 28 patients with manifest diabetes mellitus or decreased glucose tolerance. Each test was evaluated with the aid of an analogue computer model, and parameters characterizing different parts of the insulin curve during GIT were obtained. Large variations existed in all parameter values both in the normal and diabetic groups, and the overlapping between the two groups was considerable. In 15 out of 85 healthy subjects the plasma insulin response during GIT was of the diabetic type as judged from the frequency distribution of the computer parameters (low values). The similarity was still more striking when the characteristics of the insulin curves in these 15 subjects were compared with those in patients with mild diabetes or with a decreased glucose tolerance only. It is postulated that this type of low insulin response reflects a derangement of the release of insulin into the circulation, and that it marks an alteration which probably is a prerequisite for the development of diabetes mellitus. In this sense, these subjects may be considered to be potential diabetics.
Article
The early phase of insulin release in the first five minutes after intravenous administration of glucose, glucagon , and glucose-plus-glucagon was investigated systematically in various clinical conditions. In normal subjects there is an immediate release of insulin after glucose, glucagon, and glucose-plus-glucagon infusions. The latter combination produced the highest insulin levels. Of a group of nonobese subjects with diabetic heritage , some had impaired early release of insulin, but0 their mean response did not differ significantly from the normal group. Investigation of nonobese potential diabetics (offspring of two diabetic parents) revealed that as a group average they had decreased insulin levels during the early phase of insulin release, even though intravenous glucose tolerance was normal. Four of ten subjects had a normal response. Nonobese, noninsulin-dependent diabetics had no insulin response to infused glucose, but when glucagon was added to glucose a significant and rapid insulin discharge was observed. However, the magnitude of this response was about half that seen in normal subjects after glucose-plusglucagon. Finally, the early phase of insulin release was studied in obese nondiabetic subjects who demonstrated an exaggerated insulin release to each stimulus. Again, glucose-plusglucagon was the most potent stimulator of insulin release. It is postulated that impairment in the early phase of insulin release may be the first detectable abnormality of insulin secretion in diabetes mellitus and that glucagon has the capability of restoring this toward normal.
Article
The effect of various inhibitors on insulin release from pieces of rabbit pancreas incubated in vitro was studied. Insulin release was stimulated by glucose (3mg./ml.), leucine (5mm), tolbutamide (200mug./ml.), ouabain (10mum), a raised extracellular K(+) concentration (60mm) and substitution of the Ca(2+) content of the incubation medium by Ba(2+) (2.5mm). (a) Mannoheptulose (6mg./ml.) inhibited glucose-stimulated insulin release only. (b) Anoxia abolished or inhibited insulin release stimulated by glucose, leucine, tolbutamide and K(+), but had little or no effect on release stimulated by ouabain or Ba(2+). (c) 2,4-Dinitrophenol (0.25mm) abolished or inhibited insulin release stimulated by glucose, ouabain or Ba(2+). (d) Diazoxide (250mug./ml.) abolished or inhibited insulin release stimulated by glucose, leucine, tolbutamide, ouabain or Ba(2+) (0.25 or 1mm). Diazoxide had no effect on insulin release stimulated by Ba(2+) (2.5mm) and potentiated release stimulated by K(+). (e) Adrenaline (1mum) abolished insulin release stimulated by glucose, leucine, tolbutamide, ouabain or Ba(2+). K(+)-stimulated release was inhibited by adrenaline. (f) Tetrodotoxin (1mum) had no effect on insulin release stimulated by glucose, leucine, tolbutamide, ouabain, K(+) or Ba(2+). (g) Nupercaine (1mm) abolished insulin release stimulated by glucose or Ba(2+).
A method for the microdissection of intact pancreatic islets of mammals
  • B Hellman
Hellman, B.: Studies in obese-hyperglycemic mice. Ann. N.Y. Acad. Sci. 13 1: 541-558, 1965. 8. Hellerstriim, C.: A method for the microdissection of intact pancreatic islets of mammals. Acta Endocr. (Kobenhavn) 45: 122-132, 1964.