Synchronization of mouse islets of Langerhans by glucose waveforms

Pancreatic islets secrete insulin in a pulsatile manner, and the individual islets are synchronized, producing in vivo oscillations. In this report, the ability of imposed glucose waveforms to synchronize a population of islets was investigated. A microfluidic system was used to deliver glucose waveforms to ～20 islets while fura 2 fluorescence was imaged. All islets were entrained to a sinusoidal waveform of glucose (11 mM median, 1 mM amplitude, and a 5-min period), producing synchronized oscillations of fura 2 fluorescence. During perfusion with constant 11 mM glucose, oscillations of fura 2 fluorescence were observed in individual islets, but the average signal was nonoscillatory. Spectral analysis and a synchronization index (λ) were used to measure the period of fura 2 fluorescence oscillations and evaluate synchronization of islets, respectively. During perfusion with glucose waveforms, spectral analysis revealed a dominant frequency at 5 min, and λ, which can range from 0 (unsynchronized) to 1 (perfect synchronization), was 0.78 ± 0.15. In contrast, during perfusion with constant 11 mM glucose, the main peak in the spectral analysis corresponded to a period of 5 min but was substantially smaller than during perfusion with oscillatory glucose, and the average λ was 0.52 ± 0.09, significantly lower than during perfusion with sinusoidal glucose. These results indicated that an oscillatory glucose level synchronized the activity of a heterogeneous islet population, serving as preliminary evidence that islets could be synchronized in vivo through oscillatory glucose levels produced by a liver-pancreas feedback loop.     

Cryopreservation of islets of Langerhans

Development of techniques for cryopreservation of pancreatic islets of Langerhans could potentially allow for increased freedom from the time restrictions presently affecting viability in islet cell transplantation. While several investigators have attempted islet cell freezing and have obtained favorable in vitro results after thawing, there have been few reported in vivo successes with islets transplanted after freezing. We have developed a simple system for freezing islet cell pancreatic fragments to ?196 °C and have either stored them in liquid nitrogen for 24 hr or immediately thawed the islets prior to transplantation. In addition, antilymphoblast globulin has been used as graft pretreatment modality in order to modify islet cell immunogenicity. We found that ALG was effective in prolongation of graft survival after freezing as well as on fresh nonfrozen transplants. The use of freezing and ALG appears, therefore, to have a favorable effect on the immunogenicity of the pancreatic islet cell allograft.     

Glucokinase in B-cell-depleted islets of Langerhans

Glucose phosphorylation was studied in B-cell-enriched or in B-cell-depleted pancreatic islets from normal or streptozotocin-diabetic rats, respectively, using quantitative histochemical procedures. The data indicate that B-cell-enriched preparations from normal animals and whole islets from normals, diabetics, and insulin-treated diabetic animals have comparable glucokinase activities. Average maximum velocities were (mmol/kg dry tissue/hr) 134.1 +/- 7.3 for whole islets and 125.6 +/- 10.7 for the B-cell-enriched preparations from normal rats, 143.1 +/- 13.6 for B-cell-depleted islets from diabetic rats, and 124.4 +/- 10.7 for B-cell-depleted islets from insulin-treated diabetic animals. The Kmax for glucose of the enzyme in islets from untreated diabetic rats was 16 mM, comparable to the Kmax found for glucokinase from normal rat islets. Mannoheptulose, previously shown to be a competitive inhibitor of glucokinase from liver and normal islets, also inhibited glucokinase in B-cell-depleted islets from diabetic rats. The data indicate that glucokinase is not selectively located in the B-cell, as was previously assumed, but is also found in A- and/or D-cells of diabetic rats. This observation raises significant questions about the functional role of islet glucokinase under control and diabetic conditions.     

Evidence for modulation of cell-to-cell electrical coupling by cAMP in mouse islets of Langerhans

The effects of forskolin on electrical coupling among pancreatic β-cells were studied. Two microelectrodes were used to measure membrane potentials simultaneously in pairs of islet β-cells. Intracellular injection of a current pulse (ΔI) elicited a membrane response ΔV₁ in the injected cell and also a response ΔV₂ in a nearby β-cell confirming the existence of cell-to-cell electrical coupling among islet β-cells. In the presence of glucose (7 mM), application of forskolin evoked a transient depolarization of the membrane and electrical activity suggesting that the drug induced a partial inhibition of the β-cell membrane K⁺ conductance. Concomitant with this depolarization of the membrane there was a marked decrease in β-cell input resistance (ΔV₂/ΔI) suggesting that exposure to forskolin enhanced intercellular coupling. Direct measurements of the coupling ratio ΔV₂/ΔV₁ provided further support to the idea that forskolin enhances electrical coupling among islet cells. Indeed, application of forskolin reversibly increased the coupling ratio. These results suggest that cAMP might be involved in the modulation of electrical coupling among islet β-cells.     

Changes in phosphate do not affect insulin release from isolated mouse islets of Langerhans

Phosphate affects glucose tolerance, insulin release and peripheral insulin sensitivity. In the present study moderate changes in the phosphate concentration of the incubation medium from 0.3 to 2 mmol/l (i.e., within the physiological range) did not affect insulin release from isolated mouse islets in vitro. In addition, the vitamin-D status of the animals had no effect on the glucose-stimulated insulin response in the different phosphate concentrations. Therefore these data indicate that the impaired glucose tolerance seen in hypophosphatemic states is not due to a direct effect of phosphorus levels on the insulin-releasing B-cell.     

Glucose-induced oscillatory changes in extracellular ionized potassium concentration in mouse islets of Langerhans.

Liquid membrane [K+]-sensitive microelectrodes (1-2 micron tip diameter) were used to measure the extracellular ionized potassium concentration in mouse pancreatic islets of Langerhans. With the tip of the microelectrode at the surface of the islet, the time course of the [K+]-sensitive electrode potential changes in response to the application of rapid changes in [K+]o (from 1.25 to 5 mM), could be reproduced by the equation for K+-diffusion through a 100-micron-thick unstirred layer around the islet (diffusion coefficient for K+ at 27 degrees C, DK,o, taken as 1.83 X 10(-5) cm2/s). The time to reach 63% of the steady-state electrode response with the tip in the chamber at the surface of the islet was from 5 to 6 s. When the tip of the [K+]-sensitive electrode was placed in the islet tissue, the time for the response to reach 63% of the steady-state level increased. The time course of the [K+]-sensitive electrode response could be reproduced using the same diffusion model assuming that K+ diffusion into the islet tissue takes place in a tortuous intercellular path with an apparent diffusion coefficient, DK,I, about half of DK,o, in series with the unstirred layer around the islet. In the absence of glucose the potassium concentration in the extracellular space, [K+]I, was found to be higher than the concentration in the external modified Krebs solution, [K+]o. The difference in concentration [K+]I - [K+]o was greater when [K+]o was smaller than 2 mM. In the presence of glucose (between 11 and 16 mM), under steady-state conditions, small oscillatory changes in the [K+], (1.48 +/- 0.94 mM) were detected. Simultaneous recording of membrane potential from one B-cell and [K+], in the same islet indicated that the potassium concentration increased during the active phase of the bursts of electrical activity. Maximum concentration in the intercellular was reached near the end of the active phase of the bursts. We propose that the space between islet cells constitutes a restricted diffusion system where potassium accumulates during the transient activation of potassium channels.     

Synthesis of glycosaminoglycans by islets of Langerhans

Incorporation of (35S)-sulfate into glycosaminoglycans (GAG) of toadfish islets of Langerhans in vitro was examined. (35S)-sulfated GAG were synthesized by a component of the microsomal fraction, and subsequently transferred to the secretion granules, mitochondria and nuclei. The predominant type of GAG synthesized was heparan sulfate, but chondroitin 4- and 6-sulfate and dermatan sulfate were also found.     

ADP-ribosylation of isolated rat islets of Langerhans

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.     

Detection of multiple patterns of oscillatory oxygen consumption in single mouse islets of Langerhans.

A novel oxygen microsensor was used to measure oxygen levels in single mouse islets as a function of glucose concentration. Oxygen consumption of individual islets was 5.99 +/- 1.17, 9.21 +/- 2.15, and 12.22 +/- 2.16 pmol/min at 3, 10, and 20 mM glucose, respectively (mean +/- SEM, n = 10). Consumption of oxygen was islet-size dependent as larger islets consumed more oxygen than smaller islets but smaller islets consumed more oxygen per unit volume than larger islets. Elevating glucose levels from 3 to 10 mM induced pronounced fast oscillations in oxygen level (period of 12.1 +/- 1.7 s, n = 6) superimposed on top of large slow oscillations (period of 3.3 +/- 0.6 min, n = 6). The fast oscillations could be completely abolished by treatment with the L-type Ca2+-channel blocker nifedipine (40 microM) with a lesser effect on slow oscillations. Slow oscillations were almost completely dependent upon extracellular Ca2+. The oxygen patterns closely mimic those that have previously been reported for intracellular Ca2+ levels and are suggestive of an important role for Ca2+ in amplifying metabolic oscillations.     

Phosphorylation reactions in islets of Langerhans.

The possible significance of phosphorylation reaction in islets of Langerhans in relation to the secretion of insulin is discussed. The secretagogues, glucose and its metabolites, cAMP and Ca++ and their influences on protein-kinase activity are given particular attention. The data obtained by the authors, as well as by other groups, are in agreement that cAMP is a potent stimulator of protein kinase activity. Glucose and its metabolites influenced protein kinase activity in one instance. Ca++ in supraphysiological amounts inhibited protein phosphorylation. The links between phosphorylation reactions and insulin secretion are, at the present time, conjectural.