Effect of Na+, K+ and Mg2+ on 45Ca+ uptake by pancreatic islets.

Microdissected pancreatic islets of noninbred ob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable 45Ca2+ uptake by islet cells. Omission of Mg2+ from the incubation medium had no effect, but the 45Ca2+ uptake was increased by omission of Na+ and decreased by omission of K+. Excess Mg2+ (1.

Metabolism of cold-stored pancreatic islets.

A previous study showed that the ability of glucose to stimulate insulin release was retained in islets stored at 8 degrees C for one week provided that glucose was present in a high concentration in the storage medium. The metabolic properties of islets stored in the cold have now been further explored in an attempt to clarify the protective effect of glucose. During storage in the cold the islet formation of 3H2O from (5--3H) glucose and oxygen consumption were only a few per cent of that of fresh islets whereas the putake of 86Rb+ was 20--48%.

Glucose inhibition of 45Ca efflux from pancreatic islets.

Pancreatic islets were microdissected from ob/ob mice, loaded for 2 h with 45Ca and perfused with calcium-deficient medium. Irrespective of the glucose and calcium concentrations in the loading medium, increased glucose in the perfusion medium resulted in reduced amounts of radioactivity in the perfusate. A glucose inhibition of 45Ca washout was also evident when the specific radioactivity of the islets approached that of the labeling medium, indicating that the effect was not simply due to isotopic dilution.

Calcium and pancreatic beta-cell function. 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools.

beta-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of 45Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37 degrees C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable 45Ca. The stimulatory effect of phosphate was additive to that produced by glucose.

Further studies on the relationship between insulin release and lanthanum-nondisplaceable 45Ca2+ uptake by pancreatic islets: effects of fructose and starvation.

Relationships between the release of insulin and the incorporation of 45Ca2+ into a lanthanum-nondisplaceable (intracellular) pool were studied in islets microdissected from the pancreatic glands of non-inbred ob/ob mice. In comparison with D-glucose, D-fructose was slowly oxidized and had only marginal effects on insulin release. However, fructose was as effective as glucose in stimulating the lanthanum-nondisplaceable 45Ca2+ uptake.

Development of the insulin secretory defect in genetically diabetic (db/db) mouse.

Genetically diabetic mice (C57BL/KsJ-db/db) were used as a model to study the development of defects of insulin secretion in relation to common metabolic indicators (body weight, serum glucose and insulin, and islet insluin contant). Consistent with the idea of a protective effect of oestrogen on the pancreatic beta-cell, the female diabetic mice survived longer than the males. In males, while serum insulin decreased in the later stages of the disease, serum glucose increased progressively with age.

Calcium and pancreatic beta-cell function. 2. Mobilisation of glucose-sensitive 45Ca from perifused islets rich in beta-cells.

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.

Calcium and pancreatic beta-cell function: glucose stimulation uptake of lanthanum-displaceable 45-Ca from low or normal calcium-containing media.

The uptake of 45Ca was studied in beta-cell-rich pancreatic islets microdissected from ob/ob-mice. Glucose stimulated 45Ca incorporation in a lanthanum-displaceable pool remaining after correction for the extracellular space occupied by sucrose. It is suggested that the glucose effect on the lanthanum-displaceable calcium is the cause rather than the result of the secretion of insulin since it could be demonstrated also in the presence of only trace amounts of extracellular calcium.

Collagenase isolation and 45Ca efflux studies of human islets of Langerhans.

22 human pancreases were removed soon after circulatory arrest from donors aged 15--63 years. Part of the pancreas was used for isolation of islets by the collagenase technique. The mean yield +/- SEM, expressed as the number of islets isolated from 4 g pancreas was 79 +/- 15.

Islet contents of cyclic 3',5'-guanosine monophosphate under conditions which affect the cyclic 3',5'-adenosine monophosphate.

The sensitivity of the radioimmunoassay for cGMP was considerably increased by previous 2'-O-succinylation of the nucleotide. The basal content of cGMP in beta-cell-rich pancreatic islets isolated from ob/ob-mice was similar to that of cAMP, i.e.

Calcium and pancreatic beta-cell function. I. Stimulatory effects of pentobarbital on insulin release.

Islets microdissected from ob/ob-mice were exposed to 3mM pentobarbital in media which were normal or deficient in Ca2+. This treatment resulted in marked decrease of the islet content of cyclic AMP recorded in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Pentobarbital had a dual effect on insulin release.

Glucose-stimulated and La3+-nondisplaceable Ca2+ pool in pancreatic islets.

To study intracellular pools of calcium tissue specimens from noninbred ob/ob mice were labeled with 45Ca2+ and subsequently washed with La3+. D-glucose, 20 mM, enhanced the labeling of the La3+-nondisplaceable calcium in pancreatic islets but not in pieces of exocrine pancreas or liver. The disappearance of 45Ca2+ from labeled islets was accelerated by the ionophore, X-537A, but not by dibutyryl cyclic AMP, theophylline, or pentobarbital.

Metabolic characteristics of pancreatic beta-cells exposed to calcium-transporting ionophores.

The effects of the ionophores A-23187 and X-537 A on glucose metabolism, ATP content and sucrose permeability in pancreatic islets microdissected from obese-hyperglycemic mice were studied. The formation of 14CO2 from 10 mM D-[U-14C] GLUCOSE WAS INHIBITED BY OMISSION OF Ca2+ from the medium. A-23187 (10 muM) induced a further decrease of 14CO2 formation whereas X-537 A (10 muM) had no effect.

Effects of various modifiers of insulin release on the lanthanum-nondisplaceable 45Ca2+ uptake by isolated pancreatic islets.

The uptake of 45Ca2+ by a lanthanum-non-displaceable pool in pancreatic islets was studied; Raising the extracellular D-glucose concentration from 3 to 20 mM stimulated the 45Ca2+ uptake in hand-dissected islets of ob/bo-mice as well as in collagenase-isolated islets of ob/ob or normal mice. The effect was dose-dependent in the range of 0-20 mM D-glucose and was seen throughout a wide range of extracellular calcium concentrations (16 mumol-2.56 mmol of Ca2+ added per litre of medium).

Calcium and secretion: distinction between two pools of glucose-sensitive calcium in pancreatic islets.

D-Glucose, but not L-glucose or 3-O-methyl-D-glucose, stimulates 45Ca2+ uptake by both lanthanum-displaceable and lanthanum-nondisplaceable pools in pancreatic islets. The nondisplaceable pool probably represents secretory granules, while the displaceable pool may be located in the beta-cell membrane. Kinetic studies with isotopically labeled islets suggest that only the displaceable pool participates in the short-term coupling of the glucose stimulus with secretion.

Effects of dextran-linked chloromercuribenzoic acid on insulin release from microdissected pancreatic islets.

Insulin release in response to dextran-linked p-chloromercuribenzoic acid was studied in microdissected pancreatic islets of non-inbred ob/ob-mice. No contamination of the dextran-linked mercurial with free chloromercuribenzoic acid was detected before or after the incubation with islets. In comparison with free mercurial, of the same thiol-blocking activity, the dextran-linked compound had a weak insulin-releasing action with a different dose vs.

Ionic effects on the uptake of sulfonylurea (glibenclamide) by pancreatic islets.

The accumulation of glibenclamide was studied in the pancreatic islets of non-inbred ob/ob-mice. Microdissected islets were incubated in media of different ionic composition and the accumulation measured as the uptake of drug not accounted for by equilibration in the urea space. In 12 mM N-hydroxyethylpiperazine-N'-2-ethane sulphonic acid (HEPES) buffer the accumulation was only half of that in Krebs-Ringer bicarbonate buffer.

On the possible role of thiol groups in the insulin-releasing action of mercurials, organic disulfides, alkylating agents, and sulfonylureas.

The thiol activity of pancreatic islets was spectrophotometrically assayed as the formation of 6-mercaptonicotinic acid from the organic disulfide, 6,6'-dithiodinicotinic acid. Islets containing more than 90% beta-cells were microdissected from non-inbred ob/ob-mice. Comparisons of intact with homogenized islets indicated that the organic disulfide penetrates relatively slowly into the beta-cells.

Effects of starvation and Ca++ on glucose-induced accumulation of cyclic 3',5'-AMP in pancreatic islets.

Exposure to glucose in the presence of 3-isobutyl-1-methylxanthine leads to accumulation of cAMP in islets microdissected from ob/ob mice. This process is dependent on extracellular Ca++ but differs markedly from the glucose action on insulin release in the same in vitro system in disappearing after 18 h of starvation.

Maintenance of insulin release from pancreatic islets stored in the cold for up to 5 weeks.

Insulin content and release were measured from hand-dissected pancreatic islets from noninbred ob/ob mice after 1-5 wk storage in tissue culture medium 199 at various temperatures and glucose concentrations. After storage of islets for 1 wk at 37 degrees, 22 degrees, or 8 degrees C in 18 mM glucose medium and preincubation with 1 mM glucose, glucose-stimulated insulin release during the subsequent incubation was only 20-35% of that of fresh islets. The addition of a 4-h period at 37 degrees C with 18 mM glucose between the cold storage and perincubation restored glucose-stimulated insulin release from 8 degrees C stored islets to fresh-islet levels.

Effects of glucose on 45Ca2+ uptake by pancreatic islets as studied with the lanthanum method.

1. Fluxes of 45Ca2+ were studied in pancreatic islets from non-inbred ob/ob-mice. Because La3+ blocked the transmembrane fluxes of 45Ca2+ in islet cells, incubations aimed at measuring glucose-induced changes of the intracellular Ca2+ were ended by washing the islets with 2 mM-La3+ for 60 min.

Influence of anoxia on glucose metabolism in pancreatic islets: lack of correlation between fructose-1,6-diphosphate and apparent glycolytic flux.

When equilibrated with O2-CO2 (95:5), pancreatic islets of non-inbred ob/ob-mice exhibited a sigmoidal dependence of 3H2O production on D-(5-3H)-glucose concentration; the rate was most sensitive to changes of glucose concentration around 5mM and tended to be maximum above about 15mM glucose. 3H2O production from more than 5 mM D-(5-3H)-glucose was about twice as fast as the production of 14CO2 from equimolar D-(U-14C)-glucose. Islets equilibrated with N2-CO2 (95:5) did not exhibit a sigmoidal dose-response curve for 3h2o production, the process being inhibited by anoxia at glucose concentrations above 5mM.