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.     

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.     

A high yield method for isolating rat islets of Langerhans using differential sensitivity to freezing

Conventional methods of isolating islets of Langerhans rely upon the differential sensitivity of the pancreatic acinar tissue vs islets to enzymatic dissociation by crude collagenase, however, the yield of intact islets obtainable with this technique is quite low. Higher yields of islets can be obtained with pharmacological or surgical methods which either destroy acinar cells or cause them to release their zymogen granules. However, because of the requirement to pretreat the donor, these methods cannot be scaled-up for potential clinical use. To overcome the limitations of the conventional isolation procedures, we exploited the differential sensitivity of acinar cells and islet cells to freezing damage. Using this approach we are able to isolate greater than 2500 islets from the pancreas of a single rat. Basically, we rapidly mince pancreatic tissue, subject the tissue to a short collagenase digestion, briefly freeze the tissue at -30 degrees C in the presence of glycerol, and immediately thaw it. Subsequent enzyme treatment digests the residual acinar tissue, collagen, DNA, and proteins. Preliminary results indicate that the islets are morphologically indistinguishable from islets isolated using conventional digestion techniques.     

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 human ovarian tissue: comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

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.     

Adventures with insulin in the islets of Langerhans

Insulin is a small but beautifully organized protein with a unique two-chain structure, the first protein to be sequenced. The mechanism of its biosynthesis invited much initial speculation but was finally clarified by the discovery of proinsulin, its single-chain precursor. The rich present-day field of protein precursor processing via post-translational proteolysis within the secretory pathway arose in the early 1970s as an offshoot of studies on insulin biosynthesis, which provided a novel paradigm for the generation of many other small neuroendocrine peptides. Before long, this mechanism was also found to play a role in the production of a much wider spectrum of proteins traversing the secretory pathway (receptors, growth factors, blood-clotting components, and even many viral envelope proteins) occurring in almost all eukaryotic cells. Indeed, yeast provided a key clue in the search for the proprotein convertases, the endoproteases that work along with carboxypeptidases and other modifying enzymes, such as the amidating enzyme complex (PAM), in converting inactive or less active precursor proteins into their fully active peptide products. In this "Reflections" article, I have tried to recount the people and events in my life that led to my involvement first in basic biochemical research and then on to insulin, proinsulin, and many relevant related areas that continue to fascinate and challenge my colleagues and me, as well as many other biomedical scientists today, as diabetes mellitus increasingly threatens human health throughout our contemporary world.     

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.     

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.     

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.     

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.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.