Pancreatic islets exposed to 11 mM glucose exhibited complex variations of cytoplasmic Ca2+ concentration ([Ca2+]i) with slow (0.3‐0.9 min−1) or fast (2‐7 min−1) oscillations or with a mixed pattern. Using digital imaging and confocal microscopy we demonstrated that the mixed pattern with slow and superimposed fast oscillations was due to separate cell populations with the respective responses. In islets with mixed [Ca2+]i oscillations, exposure to the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (DTBHQ) resulted in a selective disappearance of the fast pattern and amplification of the slow pattern. In addition, the protein kinase A inhibitor RP‐cyclic adenosine 3′,5′‐monophosphorothioate sodium salt transformed the mixed [Ca2+]i oscillations into slow oscillations with larger amplitude. Islets exhibiting only slow oscillations reacted to low concentrations of glucagon with induction of the fast or the mixed pattern. In this case the fast oscillations were also counteracted by DTBHQ. The spontaneously occurring fast oscillations seemed to require the presence of cAMP‐elevating glucagon, since they were more common in large islets and suppressed during culture. Image analysis revealed [Ca2+]i spikes occurring irregularly in time and space within an islet. These spikes were preferentially observed together with fast [Ca2+]i oscillations, and they became more common after exposure to glucagon. Both the slow and fast oscillations of [Ca2+]i in pancreatic islets rely on periodic entry of Ca2+. However, the fast oscillations also depend in some way on paracrine factors promoting mobilization of Ca2+ from intracellular stores. It is proposed that such a mobilization in different cells within a tightly coupled islet syncytium generates spikes which co‐ordinate the regular bursts of action potentials underlying the fast oscillations.

Calcium-magnesium interactions, total amounts of intracellular magnesium, and insulin release were studied in beta-cell-rich pancreatic islets from ob/ob mice. Mg2+ inhibited the uptake of intracellular 45Ca and insulin release induced by glucose or high concentrations of potassium. Omission of Mg2+ from a Ca2+-deficient medium resulted in an increased efflux of 45Ca, whereas the characteristic glucose inhibition of the efflux was diminished. After addition of Mg2+ to a Mg2+-depleted medium, the glucose-stimulated 45Ca efflux was markedly reduced. Mg2+ inhibited the basal efflux of 45Ca, and this effect was preceded by a transient stimulation. Ca2+ but not Mg2+ stimulated 45Ca efflux in a medium depleted of Ca2+, Mg2+, and Na+. The data indicate that Mg2+ interferes with Ca2+ entry through voltage-dependent Ca2+ channels. Mg2+ may also inhibit the outward transport of Ca2+ from the cells at a site different from the Na+-Ca2+ countertransport mechanism. The total amount of intracellular magnesium remained unaffected by glucose and was not changed unless the ionic composition of the mediums were changed grossly. Under physiological conditions it is therefore unlikely that fluctuations in the intracellular Mg2+ concentration are part of the mechanism by which the functionally important Ca2+ is regulated.

The effects of N iodoacetyl 2 amino 2 deoxy (D) glucose and various N bromoacetylglycosylamines on the release of insulin from microdissected pancreatic islets of non inbred ob/ob mice were studied. N Bromoacetyl β (D) glucosylamine (10 m(M)) initiated insulin release in the absence of (D) glucose and, at concentrations of 2.5-10 m(M), but not 20 m(M), potentiated insulin release in response to 10 m(M) (D) glucose. The potentiating, but not the initiating, action was significantly inhibited in the presence of mannoheptulose. N Bromoacetyl β (L) glucosylamine or N bromoacetyl β (D) galactosylamine had no effect in the absence of (D) glucose. However, 2.5-20 m(M) concentrations of the (L) glucose derivative and 1.25-5.0 m(M) concentrations of the (D) galactose derivative potentiated the effect of 10 m(M) (D) glucose; at 20 m(M) the (D) galactose derivative inhibited the (D) glucose induced insulin release. N Iodoacetyl 2 amino 2 deoxy (D) glucose (0.1-10 m(M)) did not initiate or potentiate insulin release but, at a concentration of 10 m(M), inhibited the effect of (D) glucose. The results support this hypothesis that alkylation of thiol groups in the β cell plasma membrane leads to potentiation of (D) glucose induced insulin release if glycolysis is not simultaneously inhibited by the thiol reagent. If a regulatory site ('direct receptor') for the (D) glucose molecule plays a role in stimulus recognition, N iodoacetyl 2 amino 2 deoxy (D) glucose may be valuable in attempts to label and isolate it. (Less)

Mice, 7-8-mo old, of the C57BL/Ks,l-db strain and homozygotic for the mutant gene db, exhibited marked hyperglycemia and moderately elevated serum insulin levels. Light and electron microscopy provided evidence of a slightly decreased proportion of ~ cells in the pancreatic islets, irregular islet architecture with intraislet ducts, and degenerative as well as hypertrophic changes in the individual cells. As a rule, islets microdissected from these mice did not release insulin in response to glucose, theophylline, iodoacetamide, or chloromercuribenzene-p-sul- phonic acid. The absence of secretory responses was not simply due to lack of insulin. Although the islet content of insulin was decreased in C57BL/KsJ-db/db mice, the remaining amount was severalfold larger than that released from stimulated islets of normal controls. Another mutation, db 2J, an allele of db with identical phenotypic expressions in the C57BL/KsJ strain, was studied on the genetic background C57BI/6J. In contrast to the severely diabetic C57BL/KsJ- db/db animals, the C57BL/6J-db2J/db ~J mice were characterized by highly elevated serum insulin levels and only moderate hyperglycemia. Their endocrine pancreas was enlarged and showed an increased proportion of ~ cells. Like the islets of normal mice, those of C57BL/6J-db~a/dbZJ mice responded to glucose and chloromercuribenzene-p-sulphonic acid, the glucose-induced responses being po- tentiated by theophylline or iodoacetamide. C57BL/KsJ-db/db mice should provide a valuable model for studying defects in insulin secretion in relation to diabetes mellitus. Mice of the C57BL/6J strain offer a control material that may help to elucidate the dependence of the insulin secretory defect on the background genome.

Treatment of pancreatic islets from ob/ob-mice with bacterial neuraminidase (0.8 to 40 mU/ml) resulted in a significant decrease of the sialic acid content and of the secretory response to glucose. The inhibitory effect on the glucose stimulated insulin release was reproduced with different batches of neuraminidase from Clostridium perfringens and Vibrio cholera. Treatment with neuraminidase affected neither the insulin content of the islets nor the potentiating effect of theophylline in the presence of 20 mM glucose. The results suggest that sialic acid plays a role in the mechanism of insulin release and that the inhibitory effect of sialic acid depletion is overcome by theophylline. (30 references.)

Phlorizin (10 mM) inhibited glucose-stimulated insulin release from microdissected pancreatic islets of obese-hyperglycemic mice. In the absence of glucose, phlorizin (5-15 mM) as well as phloretin (10 mM) stimulated insulin release. These stimulatory effects were inhibited by mannoheptulose, suggesting that phlorizin and phloretin were sensed by the system which recognizes glucose as an insulin secretagogue. However, the mechanism sensitive to phlorizin does not seem to possess the full competence of the glucose-recognizing system, since phlorizin did not potentiate the insulin-releasing actions of arginine or theophylline. Leucine, but not pyruvate or succinate, enhanced the stimulatory effect of phlorizin. Radioactive phlorizin rapidly accumulated in amounts far exceeding the urea space of the islets. This uptake was concentration-dependent up into the millimolar concentration range. It was not significantly influenced by glucose. Antimycin A, p-chloromercuriphenyl-sulfonic acid, and chlorpromazine, which increase the uptake of extracellular space markers, stimulated the uptake of phlorizin in whole islets but not in islet homogenates. It is suggested that phlorizin binds predominantly to the plasma membranes of intact β-cells. Although the binding may not be specific for glucose sites, reaction with such a site could be responsible for the phlorizin-induced insulin release.

To elucidate the role of biogenic amines in insulin secretion, pancreatic islets rich in β-cells were microdissected from obese-hyperglycemic mice and were incubated with 14C-labelled 5-hydroxytryptamine (5-HT). The saturability of uptake and the fact that 5-HT was accumulated to high levels indicated that the β-cells possess a transport system with great capacity for this amine. The initial uptake was not sensitive to glucose or diazoxide.

Free amino acids were measured with a microtechnique employing labelling with 14C-dansyl-Cl and separation with two-dimensional thin-layer chromatography. Most amino acids occurred in greater amounts in the islets than in the exocrine pancreas. Aspartic acid, valine and leucine were present in tenfold amounts. It is postulated that the β-cells are equipped with binding sites for large amounts of the latter amino acid. Like nervous tissue, the pancreatic islets were characterized by the presence of gamma-aminobutyric acid and high levels of taurine.

Microdissected pancreatic islets of obese-hyperglycemic mice were used to study the uptake of mannoheptulose by β-cells in relation to the dynamics of mannoheptulose-inhibited insulin release. Mannoheptulose uptake was faster at 37° than at 8° and was inhibited by glucose, 3- O -methylglucose, or phlorizin. The transport rate for mannoheptulose was much lower than that previously observed for glucose. Nevertheless, a few minutes of exposure to 5 mM mannoheptulose were enough to establish intracellular concentrations of the same magnitude as the concentrations inhibiting insulin release and glucose oxidation when added to the medium. This observation makes it possible to explain the prompt inhibition of insulin release noted in microperfusion experiments as due to mannoheptulose interference with the β-cell metabolism of glucose. The mediated and glucose-sensitive transport of mannoheptulose, however, is also compatible with the idea that insulin release is governed by the binding of sugar to a receptor at the β-cell plasma membrane.

The insulin-releasing ability and uptake characteristics of non-metabolizable, transport-specific amino acids were studied in an in vitro system, using microdissected pancreatic islets with more than 90% β-cells.