Rate Of Insulin Release Research Articles

Beta-cell cytoplasmic Ca2+ balance as a determinant for glucose-stimulated insulin release.

The introduction of new techniques and the access to clonal lines of insulin-secreting cells have enabled re-evaluation of glucose effects on Ca2+ movements in pancreatic beta cells. It became evident that glucose, in addition to stimulating the entry of Ca2+, also promotes active sequestration of the ion in intracellular stores and its extrusion from the beta cells. The balance between these processes will determine the activity of Ca2+ in the cytoplasm and consequently the rate of insulin release. With the demonstration that glucose can not only increase but also lower cytoplasmic Ca2+, it follows that exposure to the sugar under certain conditions results in a paradoxical inhibition of insulin release. In diabetic patients this may be manifest as prompt reduction of circulating concentrations of insulin and C-peptide after an intravenous injection of glucose. The concept of the dual action of glucose might aid in explaining a number of poorly understood phenomena, such as the induction of rhythmic oscillations of the membrane potential of beta cells and the fact that their secretory response is improved by prolonged exposure to glucose and after priming with the sugar.

Organ culture of human foetal pancreas: conditions which affect basal and stimulated insulin release.

Human foetal pancreas has been maintained in organ culture with net synthesis and release of insulin for up to 60 days. The age of the donor foetus affected the basal insulin release rate. A plateau of secretion was reached with foetuses of greater than or equal to 16 weeks of gestation. Explants cultured within 2 h of expulsion following prostaglandin induced termination secreted 3.0 times more insulin after 20 days of culture than those cultured within 2-4 h and 8.1 times more than those cultured more than 4 h post-termination. A high oxygen environment was toxic to the explants during culture. Fresh tissue responded to a high concentration of glucose (19.3 mM) with a small but significant increase in insulin secretion. The addition of 10 mM theophylline caused a major increase in insulin release. Cultured tissue did not respond to glucose alone but did not show increased insulin release following stimulation with glucose (22 mM) together with theophylline (10 mM) in static incubation. The culture of human foetal pancreatic tissue may be useful in maintaining responsive beta cells and may help to ensure an adequate amount of donor tissue for future transplantation into diabetic patients.

Enflurane and Adenosine 3???,5???-Cyclic Monophosphate Metabolism in Pancreatic Islets

The effects of enflurane at a concentration of 4% by volume in the gas phase upon rates of insulin secretion were correlated with islet contents of adenosine 3',5'-cyclic monophosphate (cyclic AMP). At this concentration, enflurane was without effect upon the basal rate of insulin release in the presence of 2 mM glucose. By contrast, the anesthetic led to 84 and 86% inhibitions, respectively, of rates of insulin secretion stimulated by 20 mM glucose and 20 mM glucose plus 1 mM theophylline. Enflurane also led to small reductions in islet cyclic AMP content under all conditions of incubation, and a statistically significant (P less than 0.02) effect of the anesthetic in lowering cyclic AMP content was seen in islets exposed to 20 mM glucose plus 1 mM theophylline. Enflurane was without effect upon the activity of the low Km cyclic AMP phosphodiesterase of the islet under the conditions employed. By contrast, the anesthetic led to significant inhibitions of both basal and fluoride-stimulated islet adenylate cyclase activity (34%, P less than 0.01 and 23%, P less than 0.005, respectively). It is concluded that under the conditions employed, enflurane leads to inhibition of islet adenylate cyclase activity and to small resultant reductions in islet cyclic AMP content. These effects appear to be of insufficient magnitude to explain completely the observed degree of insulin secretory inhibition by the anesthetic.

Microencapsulation and controlled release of insulin from polylactic acid microcapsules.

Insulin has been encapsulated in biodegradable polylactic acid microcapsules and non-biodegradable ethylcellulose microcapsules by using an emulsification-solvent evaporation process. Gelatin and polyvinylalcohol were used as protective colloids. The concentrations and types of protective colloids affecting the micromeritic properties and release behavior of insulin microcapsules were studied. The higher the concentration of protective colloids the smaller the particle size of microcapsules. The median diameter of microcapsules decreased with the increase of the viscosity of protective colloids. Scanning electron microscopic observations suggested that microcapsules prepared from higher concentrations of polyvinylalcohol solution resulted in a nonporous and compact surface on the microcapsules, compared to the porous microcapsules prepared from gelatin solution. The residual crystals and porous structure of microcapsules affected the release rate of microcapsules. After the initial burst effect the release rate of insulin from microcapsules was found to be constant, so that prolonged release was obtainable. Three percent of polyvinylalcohol was the best choice for the preparation of polylactic acid microcapsules.

Evidence for glucose stimulation of intracellular buffering of calcium in the pancreatic beta-cell.

Glucose-induced movements of Ca2+ in pancreatic beta-cells were analysed using islets isolated from ob/ob mice and insulin-releasing cells from a clonal cell line (RINm5F). Addition of glucose to a perifusion medium resulted in an inhibited efflux of 45Ca from the islets both when the extracellular Ca2+ concentration was lower or higher than that in the cytosol. Glucose inhibition of 45Ca efflux was seen also after altering the Na+ gradient across the plasma membrane provided that the cytosolic K+ activity was maintained. The glucose suppression of 45Ca efflux corresponded to a stimulated net uptake of Ca2+ in the RINm5F cells. Also in this case the glucose effect was maintained when Na+ was replaced with K+ and suppressed after substitution with choline. During perifusion of islets with a Ca2+-deficient medium the removal of glucose resulted in a temporary stimulation of insulin release. The results suggest that glucose, in addition to stimulating the entry of Ca2+, also promotes active sequestration of the ion in intracellular stores. The balance between these processes will determine the activity of cytosolic Ca2+ and consequently the rate of insulin release.

Bombesin stimulates insulin secretion by a pancreatic islet cell line.

The amphibian tetradecapeptide, bombesin (BBS) has been shown to stimulate insulin secretion both in vivo and by pancreatic islet cells in vitro. To determine whether BBS can act directly on pancreatic beta cells, we examined its effects on insulin secretion by HIT-T15 cells (HIT cells), a clonal islet cell line. Addition of 100 nM BBS to HIT cells stimulated insulin release 25-fold within 30 sec. The rapid stimulatory effect of BBS on insulin release was short-lived: the secretory rate returned to basal levels after 90 min of BBS treatment. The decrease in the rate of insulin release in the continued presence of BBS was due not to depletion of intracellular insulin stores but to specific desensitization to this peptide. Stimulation of insulin secretion by BBS was dose dependent with an ED50 value (0.51 +/- 0.15 nM) similar to the concentration of BBS-like immunoreactive material in rat plasma. Five BBS analogs, including porcine gastrin-releasing peptide, were as powerful as BBS in stimulating insulin release. The relative potencies of the analogs tested indicated that the COOH-terminal octapeptide sequence in BBS was sufficient for stimulation of release. In contrast, 14 peptides structurally unrelated to BBS did not alter insulin secretion. BBS action was synergistic with that of glucagon; insulin secretion in the presence of maximal concentrations of both peptides was greater than the additive effects of the two peptides added individually. Somatostatin inhibited BBS-stimulated release by 69 +/- 1% with an ID50 value of 3.2 +/- 0.3 nM. These results show that BBS stimulation of insulin secretion by a clonal pancreatic cell line closely parallels its effects in vivo and support the hypothesis that BBS stimulates insulin secretion by a direct effect on the pancreatic beta cell. The clonal HIT cell line provides a homogeneous cell preparation amenable for studies on the biochemical mechanisms of BBS action in the endocrine pancreas.

Cytotoxic effects of alloxan treatment in vitro on monolayer cultures of neonatal rat pancreas.

Monolayer cultures of neonatal rat pancreas were exposed briefly (5 min) to alloxan and were then maintained for several days (up to 14 days) to examine the long-term effects of alloxan exposure on B cells. Alloxan (0-10 mM) caused a dose-dependent reduction in rates of insulin release during the first 24 h after treatment. This reduction persisted throughout the period of observation. Rates of glucagon release were unaltered by all concentrations of alloxan tested. Spontaneously decomposed alloxan had little or no effect on rates of insulin release. The simultaneous presence of high concentrations of glucose during exposure of cells to alloxan exerted a rapid protective effect against alloxan toxicity that was both glucose and alloxan dose dependent. Alloxan treatment of pancreatic monolayer cultures may allow for a comparison of those actions of alloxan that are directly on and specific for islet B cells. Furthermore, alloxan-treated cultures may represent a unique in vitro system for studying the physiology and biochemistry of other islet cell types in cultures in which B cells and insulin are reduced, as well as for studying glucose interactions with the B cell.

Activation, but not inhibition, by glucose of Ca 2+-dependent K + permeability in the rat pancreatic B-cell

The effect of glucose on the Ca 2+-activated K + permeability in pancreatic islet cells was investigated by measuring the rate of 86Rb efflux, 45Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in 86Rb outflow, 45Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased 86Rb and 45Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in 86Rb and 45Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca 2+ of the Ca 2+-sensitive modality of K + extrusion. On the contrary, as a result of its effect upon Ca 2+ handling, glucose stimulates the Ca 2+-activated K + permeability.

Immunoglobulin from insulin-dependent diabetic children inhibits glucose-induced insulin release.

Immunoglobulin was separated from islet cell antibody positive plasma of six children with newly diagnosed insulin-dependent (type I) diabetes mellitus. The dynamics of insulin release in response to glucose and partially purified antibodies were determined in dispersed rat islet cells perifused on small columns of Biogel P-2 beads. After perifusion at 5.5 mmol/L D-glucose in the presence of healthy control immunoglobulin, the rate of insulin release increased in a biphasic manner after stimulation with 30 mmol/L D-glucose. In cells exposed to diabetic immunoglobulin, 30 mmol/L D-glucose had little, if any, effect on insulin release. These results suggest that islet cell antibodies in insulin-dependent diabetes may interfere with the insulin release mechanisms in the pancreatic B-cells.

Microencapsulation of crystalline insulin or islets of Langerhans: an insulin diffusion study.

Microcapsules containing insulin crystals or islets of Langerhans were made by extruding a mixture of insulin crystals or islets and sodium alginate into a calcium chloride solution, and then coating it with poly-l-lysine. When these microcapsules were incubated at 37 degrees C, insulin could be detected readily in the medium, indicating that the microcapsular membrane is permeable to insulin. The efficiency of insulin encapsulation with crystalline insulin declined as the concentration in the sodium alginate mixture increased. Over 90% of the entrapped insulin was released after 3 days of incubation at 37 degrees C, indicating that the rate of insulin release from the microcapsules requires modification if the microcapsules are to be used as a long-term insulin delivery system. The amount of insulin secreted by the encapsulated islets was not significantly different from that of unencapsulated islets, suggesting the islets were not affected by the modified encapsulation process.

A functional analysis on isolated rat islets of Langerhans: Effects of dimethylsulfoxide and low-temperature preservation

Cryopreservation of pancreatic islets of Langerhans offers the possibility of storage of sufficient quantities of this tissue for transplantation in the treatment of certain forms of diabetes, as well as providing a means of precise histocompatibility matching. In these studies, the effects of dimethylsulfoxide and various cooling rates on islet function are examined. These studies demonstrate that islets treated with 1.4 M dimethylsulfoxide and slowly cooled at a rate of 0.3 °C/min release insulin biphasically upon glucose challenge. In addition, this stimulated release is significantly improved ( P < 0.05) by increasing the duration of post-thaw culture. After thawing, these cryopreserved islets also retain the capacity to synthesize insulin. Islets frozen at faster cooling rates (3, 14, and 48 °C/min) exhibit varying degrees of glucose-induced insulin release, indicative of freeze-induced damage. These manifestations of freeze-induced damage include high basal (nonstimulatory) insulin release rates, little or no increase in the stimulated rate versus the nonstimulated rate, and failure of the stimulated release to return to basal levels when the glucose concentration is reduced.

Influence of physical training on insulin release and glucose utilization by islet cells and liver glucokinase activity in the rat.

The effect of physical training on insulin release and glucose utilization by perifused islets and on liver glucokinase activity was examined in rats that exercised spontaneously by running (in wheel cage) up to 4-6 mi/day for 36 +/- 4 days and in sedentary controls kept in standard cages. Perifusion of islets with 4 mM glucose resulted in comparable rates of insulin release from islets obtained from trained and sedentary control rats. In contrast, when the perifusion glucose concentration was raised to 10 mM, the biphasic increase in insulin release was 40-50% lower in the trained rats as compared with untrained rats. This decrease in glucose-stimulated insulin release occurred in the face of comparable rates of glucose utilization by islets from control and trained rats. Glucose phosphorylation by liver homogenates from trained rats was reduced at all concentrations of glucose examined (0.5-100 mM). The calculated glucokinase activity was diminished by 40%, whereas hexokinase activity was decreased by 15% in the livers from trained rats. We conclude that 1) hypoinsulinemia induced by exercise training is due to decreased sensitivity of the beta-cell to the stimulant action of glucose independent of changes in islet cell utilization of glucose, and 2) exercise training results in a diminution of liver glucokinase activity that may be a consequence of the hypoinsulinemia.

Low molecular weight pineal and cerebral fractions affecting insulin secretion in vitro

Studies were performed to examine the effects of various fractions of pineal gland origin upon rates of immunoreactive insulin release during short-term incubations of pancreatic islets from pinealectomized rats. Fractions of cerebral cortex were employed in control incubations. An ultrafiltrate of pineal extracts containing materials of molecular weight ⩽ 1000 daltons stimulated insulin release while fractions of greater molecular weight were without effect. The stimulation of insulin release observed with the lower molecular weight pineal fraction was seen with both nonstimulatory (2 mM) and stimulatory (10 and 30 mM) medium glucose concentrations, but was abolished in the presence of 2,4-dinitrophenol (200 μM). Upon further fractionation of the low molecular weight pineal extract, fractions I (estimated molecular weight range 700–1000 daltons) and II (estimated molecular weight range 180–700 daltons) exhibited comparable stimulatory effects upon islet insulin release; similar effects were observed with cerebral cortical fractions in these molecular weight ranges. However, pineal fraction III (estimated molecular weight range <180 daltons) exhibited a striking inhibitory effect upon rates of insulin release compared to cerebral cortical fraction III. Potassium, dopamine, norepinephrine and epinephrine concentrations in both pineal and cortical tissue fractions were similarly low. We conclude that insulinotropic constituents) of similar molecular weight occur in pineal gland and cerebral cortex, and that the pineal insulinotropic activity stimulates active insulin secretion by the islets independently of concomitant stimulation by glucose; however, lower molecular weight insulinostatic constituents) are notable only in the pineal. It is suggested that mild hyperinsulinemia seen in pinealectomized rats under some circumstances may occur as a result of loss of the pineal insulinostatic factor(s).

Efflux of radioactive nucleotides from mouse pancreatic islets prelabelled with 2-3H-adenosine.

Cultured mouse pancreatic islets were prelabelled with 2-3H-adenosine in order to monitor the efflux pattern of radioactivity and insulin. The outflow of radioactivity decreased continuously when the islets were perifused with glucose (1.67 mmol/l). When raising the glucose concentration to 16.7 mmol/l, there was a prompt inhibition of the radioactive efflux concomitant with an increased rate of insulin release. These effects were reversed when the high glucose challenge was withdrawn. Similar radioactive efflux patterns were obtained after addition of alpha-ketoisocaproic acid, leucine or pyruvate to the perifusion medium, and also when the islets were challenged with high glucose concentrations in the absence of calcium. Both antimycin A and glipizide stimulated the efflux of radioactivity, although only the addition of glipizide was accompanied by a stimulation of the insulin release. Nucleotides constituted approximately 90% of the total effluent radioactivity. Decrease in the radioactive AMP and ADP efflux due to high glucose was furthermore found to be the cause of the observed inhibition of the total radioactive efflux. The changes in radioactive efflux induced by glucose probably reflect changes in the intracellular concentrations of AMP and ADP. It is concluded that no simple correlation exists between radioactive efflux and insulin release and that changes in the intracellular concentrations of nucleotides may be an early event in the stimulus-secretion coupling of glucose-induced insulin release.

Effects of Enflurane on Release of Insulin by Pancreatic Islets in Vitro

The effects of enflurane upon rate of insulin release from rat pancreatic islets were determined in vitro. A dose-related inhibitory effect of enflurane on glucose-stimulated insulin release was observed with almost complete inhibition being seen when the enflurane concentration in the gas phase was 3.21% (v/v), equivalent to 1.26 mM enflurane in the liquid phase. The onset on enflurane-induced inhibition was rapid, being fully developed within 5 minutes of islet exposure to the agent while the effect was equally rapidly reversed on removal of enflurane from the medium. As glucose metabolism is known to be required for glucose-stimulated insulin secretion to occur, rates of islet metabolism of [U-14C] glucose to 14CO2 were examined but these were unaffected by concentrations of enflurane up to 2.21 mM. To investigate further the metabolic integrity of islets, rates of islet incorporation of L-[4,5-3H] leucine were determined; these too were not significantly altered by concentrations of enflurane up to 1.26 mM. It is concluded that enflurane in the concentration range used clinically leads to a rapid, reversible inhibition of rat pancreatic islet insulin release, which is not attributable to interference with islet glucose metabolism or protein biosynthesis.

Regulation of glucose metabolism in pancreatic islets.

We evaluated the possible role of islet glucokinase in controlling the rate of islet glucose metabolism, and thereby the rate of glucose-induced insulin release. The activities of glucokinase, hexokinase, P-fructokinase, and glyceraldehyde-P dehydrogenase were quantitated in sonicated or isotonically homogenized islet preparations using pyridine nucleotide-dependent fluorometric assays. In sonicates, about 1/4 of the islet glucose phosphorylating activity was due to an enzyme with kinetic properties similar to glucokinase; 3/4 of the activity was due to hexokinase. The procedure for determining islet glucokinase activity was improved by centrifuging isotonic islet homogenates at 12,000 x g. The supernatant fraction was enriched for glucokinase. About 1/2 of the glucose phosphorylating activity in this fraction was due to glucokinase and 1/2 was due to hexokinase. The glucokinase activity in islet homogenates was !23 of the activity of hexokinase, 1/40 of the activity of P-fructokinase, and 1/400 of the activity of glyceraldehyde-P dehydrogenase. Detailed concentration dependency curves of glucose and mannose utilization were also obtained with intact isolated pancreatic rat islets. Glucose and mannose usage in islets was governed by two superimposed hyperbolic systems differing in Km and Vmax. A high Km system (Km for glucose 11 mM and for mannose 21 mM) predominated. A low Km system (Km for glucose 215 and for mannose 530 microM) contributed about 15% to the total activity. The available data with intact islets could be rationalized by the existence of two distinct hexose phosphorylating enzymes with differing capacities and kinetic properties. These enzymes, tentatively identified as glucokinase and hexokinase, could coexist in the same cell or could be distributed among different cell types. The possible physiologic significance of these results is discussed, emphasizing the idea of dual control of glycolysis and insulin release by glucokinase and hexokinase. An earlier proposal that glucokinase serves as glucoreceptor of beta-cells [J. Biol. Chem. 243:2730 (1968)] is greatly strengthened by the present studies.

Insulin release. Demonstration of a priming effect of 3-isobutyl-1-methyl-xanthine (IBMX) on islets of Langerhans.

Exposure of isolated rat pancreatic islets to 1 mM 3-isobutyl-1-methyl-xanthine (IBMX) resulted in a 5.7-fold increase in the rate of insulin release. After a 30-min rest period under basal conditions, reexposure of the islets to 1 mM IBMX resulted in an enhanced rate of release when compared with either a timed control -islets simultaneously exposed to IBMX for the first time-or with the first priming response to IBMX. The possibility of a continuous priming effect was suggested by the gradually rising rate of insulin release during exposure to IBMX. It is concluded that islets have a memory for IBMX exposure that results in increased responsiveness to subsequent stimulation by IBMX.

Insulin secretion induced by glucose and by stimulation of beta 2 -adrenoceptors in the rat. Different sensitivity to somatostatin.

The effects of somatostatin on insulin secretion stimulated by glucose or by the selective beta 2-adrenoceptor agonist terbutaline were studied in vivo in the anaesthetized rat. Infusion of low doses of glucose (5 mg/min) or terbutaline (2 microgram/min) caused slight stimulation of insulin secretion, whereas infusion of higher doses of glucose (12.5 mg/min) or terbutaline (200 microgram/min) yielded higher rates of insulin release. In both instances plasma insulin concentrations were of comparable magnitudes immediately prior to somatostatin infusion. Somatostatin (0.1 microgram/min) inhibited the insulin response to glucose and terbutaline, both at the low and high secretory rates. However, the inhibitory effect of somatostatin was much more pronounced on insulin release during glucose infusion than during infusion of terbutaline. Thus, at the high rate of insulin secretion somatostatin depressed plasma insulin by 46% during glucose and by 22% during terbutaline infusion. The results at the low rate insulin secretion were 66% and 48%, respectively. Both at high and low secretory rates somatostatin depressed plasma insulin levels more potently during glucose infusion than during terbutaline infusion (p less than 0.01 and P less than 0.05, respectively). Furthermore, the plasma insulin levels during inhibition by somatostatin following terbutaline stimulation stabilized after approximately 10 min of somatostatin infusion, whereas following glucose stimulation the insulin levels continued to decline. The results suggest that somatostatin inhibits insulin secretion via mechanisms that are more closely related to the insulin secretory pathway induced by glucose than to that induced by beta-adrenoceptor agonists.

The effect of physical training on insulin secretion of rat pancreatic islets.

Rats were either physically trained by a 12-wk swimming program or were freely eating or weight matched, sedentary controls. Islets of Langerhans were isolated and incubated in various glucose concentrations. Within the range of physiological glucose concentrations the rate of insulin release from islets of trained rats was lower than that from islets of sedentary controls. The DNA content of the islets was similar in the different groups. The demonstrated decreased glucose sensitivity of the insulin secretory mechanism within the beta-cells of trained rats may partly explain the finding of lower plasma insulin concentrations during intravenous glucose tolerance test in these rats compared with sedentary rats. Epididymal fat pads of trained rats were smaller than those of weight matched controls which in turn were smaller than those of freely eating controls, these differences being due to differences in fat cell size. The lower glucose sensitivity of the beta-cells in trained rats was probably not a consequence of the low body weight and small fat depots in these rats

Diabetogenic effects of chronic oral cadmium adminstration to neonatal rats.

1 Chronic exposure of neonatal rats to oral cadmium (Cd) (0.1 and 1.0 micrograms/g daily for 45 days) disturbed glucose homeostasis, as reflected by hyperglycaemia, reduced liver glycogen and enhanced gluconeogenic potential of hepatic tissue. 2 This Cd-exposure regimen also increased hepatic cyclic adenosine 3',5'-monophosphate (cyclic AMP) which was accompanied by enhancement of basal, adrenaline and glucagon-stimulated form(s) of adenylate cyclase. 3 In order to assess the responsiveness of pancreatic beta cells to glucose, islets isolated from control as well as Cd-exposed animals were incubated in vitro and their rate of insulin secretion determined. In the presence of glucose 0.5 mg/ml, there was no significant difference in the rate of insulin release. However, at higher glucose concentrations (1.5 and 3.0 mg/ml), the islets from Cd-exposed rats released significantly less insulin than those of control animals. 4 The results are discussed in relation to the possible mechanism of the diabetogenic effect of Cd.