Size distribution of mouse Langerhans islets

Pancreatic beta-cells are clustered in islets of Langerhans, which are typically a few hundred micrometers in a variety of mammals. In this study, we propose a theoretical model for the growth of pancreatic islets and derive the islet size distribution, based on two recent observations: First, the neogenesis of new islets becomes negligible after some developmental stage. Second, islets grow via a random process, where any cell in an islet proliferates with the same rate regardless of the present size of the islet. Our model predicts either log-normal or Weibull distributions of the islet sizes, depending on whether cells in an islet proliferate coherently or independently. To confirm this, we also measure the islet size by selectively staining islets, which are exposed from exocrine tissues in mice after enzymatic treatment. Indeed revealed are skewed distributions with the peak size of approximately 100 cells, which fit well to the theoretically derived ones. Interestingly, most islets turned out to be bigger than the expected minimal size (approximately 10 or so cells) necessary for stable synchronization of beta-cells through electrical gap-junction coupling. The collaborative behavior among cells is known to facilitate synchronized insulin secretion and tends to saturate beyond the critical (saturation) size of approximately 100 cells. We further probe how the islets change as normal mice grow from young (6 weeks) to adult (5 months) stages. It is found that islets may not grow too large to maintain appropriate ratios between cells of different types. Our results implicate that growing of mouse islets may be regulated by several physical constraints such as the minimal size required for stable cell-to-cell coupling and the upper limit to keep the ratios between cell types. Within the lower and upper limits the observed size distributions of islets can be faithfully regenerated by assuming random and uncoordinated proliferation of each beta-cell at appropriate rates.     

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.     

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.     

Effect of glucose on polyphosphoinositide metabolism in isolated rat islets of Langerhans.

The metabolism of inositol-containing phospholipids during insulin secretion was studied in rat islets of Langerhans preincubated with [3H]inositol to label their phospholipids. Glucose (20 mM) caused a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and an accumulation of inositol trisphosphate and inositol bisphosphate. This effect was maximal at 60s, did not require the presence of extracellular Ca2+, and was abolished by mannoheptulose (15 mM), but not by noradrenaline (1 microM). Mannose (20 mM) and DL-glyceraldehyde (10 mM) produced similar effects to those of glucose, but galactose (20 mM) and KCl (30 mM) were without effect. These results are compatible with the hypothesis that an early event in the stimulus-secretion coupling mechanism in the pancreatic B-cell is the rapid breakdown of polyphosphoinositides catalysed by phospholipase C. Moreover, they suggest that the breakdown of polyphosphoinositides is linked to sugar metabolism in the B-cell. This observation is important, since it demonstrates that events in a cell other than plasma-membrane receptor occupancy can promote polyphosphoinositide hydrolysis.     

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.     

Enzymes of glucose metabolism in normal mouse pancreatic islets

1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP⁺-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K<sub>m</sub> 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K<sub>m</sub> for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K<sub>m</sub> 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K<sub>m</sub> values of 2.5 and 21μm for NADP<sup>+</sup> and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K<sub>m</sub> values of 4 and 22μm for NADP<sup>+</sup> and glucose 6-phosphate. The K<sub>m</sub> for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP<sup>+</sup> and apparently mixed with respect to glucose 6-phosphate. 6. NADP<sup>+</sup>–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.     

Potassium-induced insulin release and voltage noise measurements in single mouse islets of Langerhans

Summary Insulin release and membrane potential fluctuations in response to increased extracellular potassium [K⁺] _o have been measured in single perifused islets of Langerhans from normal mice. An increase in [K⁺] _o from 5mm to 50mm induced a transient insulin release with a peak at about 1 min. The peak value was [K⁺] _o -dependent but the half-timet _1/2 for the decline was constant at nearly 1 min. 2.5mm cobalt completely inhibited the potassium-induced stimulation of insulin release. The insulin release elicited by 28 and 50mm [K⁺] _o was similar in terms of peak, total release and half-time from maximum release. Stepwise increase in [K⁺] _o from 10 to 28 to 50mm resulted in a normal response to 28mm but no peak of release after the 28 to 50mm increase. The results indicate good correlation between excess voltage noise, thought to reflect calcium channel activity, and insulin release evoked by changing extracellular potassium.     

Vitrification of human islets of Langerhans

Cryopreservation of human islets of Langerhans by vitrification was studied. Isolated islets were divided into four groups: (1) control islets which were cultured for 6 days, (2) islets which were vitrified after 2 days of culture, (3) control islets which were cultured for 10-13 days, and (4) islets which were vitrified after 6-9 days of culture. After warming, islets from groups 2 and 4 were cultured for 4 days. The thus treated islets were investigated with respect to insulin secretion in the presence of 2.5 or 25 mM glucose, capacity to survive during postwarming culture, and morphology. The insulin secretion in islets from all groups could be stimulated by an increase of the concentration of glucose from 2.5 to 25 mM. No significant differences were observed between the insulin secretions of the vitrified and control islets or between the islets vitrified after 2 and 6-9 days of culture. It is concluded that human islets of Langerhans cryopreserved by vitrification are functional in vitro.     

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.