Cryopreservation of mouse pancreatic islets: effects of different glucose concentrations in the post-thaw culture medium on islet recovery

It was the aim of this study to investigate the influence of the glucose concentration of the post-thaw culture medium on islet B-cell survival after cryopreservation by the combined assessments of islet recovery, islet DNA and insulin contents, and insulin release. Collagenase isolated mouse islets were kept in culture for 3 days in the presence of 11.1 mM glucose and then transferred to freezing ampoules containing Hanks' solution supplemented with 10% calf serum and 2 M dimethyl sulfoxide. After a 20-min incubation at 0 degrees C the islets were cooled at a rate of 25 degrees C/min to -70 degrees C and subsequently plunged into liquid nitrogen. After 2 hr the frozen islets were rapidly thawed at 37 degrees C, transferred to culture dishes, and cultured for another 3 days in the presence of 2.8, 5.6, 11.1, 16.7, or 28 mM glucose. Nonfrozen control islets were treated identically after a preceding 3-day culture at 11.1 mM glucose. The percentage recovery of cryopreserved islets was decreased compared to that of nonfrozen islets, but was increased when higher glucose concentrations were used in the post-thaw culture medium. Since the DNA content of the cryopreserved islets was slightly decreased, the overall survival rate of the cryopreserved B-cells, when cultured at the higher glucose concentrations after thawing, was found to be about 75%. The insulin content of the cryopreserved islets was decreased but the glucose-stimulated insulin release was essentially the same as that of the nonfrozen islets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of RNA metabolism in relation to insulin production and oxidative metabolism in mouse pancreatic islets in vitro

This study was undertaken to investigate the long-term effects of different substrates, in particular glucose, on the regulation of islet RNA metabolism and the relationship of this regulation to the metabolism and insulin production of the islet B-cell. For this purpose collagenase-isolated mouse islets were used either in the fresh state or after culture for 2 or 5 days in RPMI 1640 plus 10% calf serum supplemented with various test compounds. Islets cultured with 16.7 mM glucose contained more RNA than those cultured with 3.3 mM glucose. Culture of islets in glucose at low concentrations inhibited glucose-stimulated RNA synthesis and this inhibitory effect was reversed by prolonged exposure to high glucose concentrations. Culture with 10 mM leucine and 3.3 mM glucose or with 10 mM 2-ketoisocaproate and 3.3 mM glucose increased the total RNA content of islets as compared to that of islets cultured with 3.3 mM glucose alone. Islets cultured with 5 mM theophylline maintained a high RNA content in the presence of 3.3 mM glucose. Theophylline also increased the islet RNA content when added together with 16.7 mM glucose, as compared to 16.7 mM glucose alone. Theophylline probably exerted this effect by decreasing the rate of RNA degradation. Changes in islet RNA metabolism showed a close correlation to changes in islet total protein biosynthesis, whereas islet (pro)insulin biosynthesis and insulin release exhibited different glucose-dependency patterns. The response of islet oxygen uptake to glucose was similar to that of islet RNA and protein biosynthesis. It is concluded that the RNA content of the pancreatic islets is controlled at the levels of both synthesis and degradation. Glucose stimulates the RNA synthesis and inhibits its degradation. Moreover, the results suggest that regulation of RNA synthesis may be mediated through islet metabolic fluxes and the cAMP system.     

Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleate

The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30 kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH 6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins.     

Vasoactive intestinal polypeptide enhances hormone content and insulin release in cultured fetal rat islets

The effect of porcine vasoactive intestinal polypeptide (VIP) on development of the biphasic insulin release response in cultured fetal rat islets was investigated. Fetal islets, 21.5 days gestational age, were cultured for 7 days in RPMI 1640 culture medium containing either 2.8 or 11.1 mM glucose adn subsequently challenged with 16.7 mM glucose in a perfusion system. Islets were exposed to VIP at a final concentration of 13.2 nM by adding the peptide to the perifusion buffer (acute exposure) or by adding it to the culture medium throughout the culture period (chronic exposure). Islet hormone and DNA contents were also quantitated at the end of the culture period. Acute exposure to VIP resulted in no alterations of the insulin release pattern after culture in the presence of either glucose concentration. However, chronic treatment of islets with 13.2 nM VIP in the presence of 2.8 mM glucose resulted in significant increases in the maximum rate of insulin release during the first phase and the total amount of insulin release during both phases. Similarly, islets cultured in the presence of 11.1 mM glucose and 13.2 nM VIP demonstrated enhanced biphasic insulin release patterns with increased maximum rate and total amount of release during both phases. The presence of VIP and 2.8 mM glucose increased islet glucagon and somatostatin contents, but islet DNA and insulin contents remained unchanged. These findings indicate that VIP plays a significant role in the in vitro development of the biphasic insulin release pattern and may be a factor controlling the maturation of the fetal islet in vivo.     

Effects of glucose on 45Ca2+ outflow, cytosolic Ca2+ concentration and insulin release from freshly isolated and cultured adult rat islets

The present study aimed at comparing the effects of glucose on ionic and secretory events in freshly isolated and 5-7 day cultured rat pancreatic islets. The capacity of glucose to provoke insulin release was severely reduced in islets maintained in culture. Whether in freshly isolated or cultured islets, glucose provoked a marked and sustained decrease in 45Ca2+ outflow from islets deprived of extracellular Ca2+. In the presence of extracellular Ca2+ throughout, the magnitude of the glucose-induced secondary rise in 45Ca2+ outflow was reduced in cultured islets. Glucose provoked a weaker increase in [Ca2+]i in islet cells obtained from cultured islets than in islet cells dissociated from freshly isolated pancreatic islets. On the other hand, the stimulatory effect of carbamylcholine on 45Ca2+ outflow was unaffected by tissue culture. Lastly, in islet cells obtained from cultured islets, the increase in [Ca2+]i evoked by K+ depolarization averaged half of that observed in control experiments. These results indicate that the reduced secretory potential of glucose in cultured pancreatic islets can be ascribed to the inability of the nutrient secretagogue to provoke a suitable increase in Ca2+ influx.     

Expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms in cells of cultured rat pancreatic islets.

We have previously investigated glucose induction of glucokinase, glucose usage and insulin release in isolated cultured rat pancreatic islets (Liang, Y., Najafit, H., Smith, R. M., Zimmerman, E. C., Magnuson, M. A., Tal, M., and Mastchinsky, F. M. (1992) Diabetes (1992) 41, 792-806). Here we studied the expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms, using the same system, i.e. isolated pancreatic rat islets immediately after isolation or cultured in the presence of 3 or 30 mM glucose for as long as 10 days. We found by immunofluorescence microscopy and Western and Northern blot analysis of islet extracts that GLUT-1 expression was induced in islet beta-cells in tissue culture both with low or high glucose present. The induction of GLUT-1 was specific to beta-cells but was not present in all beta-cells and was not detected in alpha-cells. GLUT-2 expression was also specific for beta-cells and was not observed in all beta-cells. Some beta-cells in culture coexpressed GLUT-1 and GLUT-2. The expression of the two glucose transporters was regulated in the opposite direction in response to glucose concentration in the culture medium. GLUT-1 was more effectively induced when glucose was low, and GLUT-2 expression was more pronounced when glucose was high in the culture media. Another difference between the two glucose transporters was that GLUT-2 expression was increased while GLUT-1 expression was decreased as culturing continued as long as 7 days. Thus, after 7 days of culture GLUT-2 expression in beta-cells was nearly the same at low and high glucose, whereas GLUT-1 was practically absent no matter what the glucose level was. In attempts to correlate GLUT-1 and GLUT-2 expression to beta-cell function glucose uptake and glucose-stimulated insulin release in fresh and cultured islets were measured. In freshly isolated islet glucose uptake was estimated to be 100-fold in excess of actual glucose use. Glucose uptake was reduced by 7-day culture to about one-third of that observed in freshly isolated islets no matter what the glucose concentration of the culture media. We conclude that in the present experimental system GLUT-1 and GLUT-2 expression and function are not closely associated with glucose usage rates or the secretory function of beta-cells.     

Chronic exposure to TPA depletes PKCalpha and augments Ca-dependent insulin secretion from cultured rat islets

The insulin secretory responses of rat islets to glucose (15 mM), 12-O-tetradecanoylphorbol13-acetate (TPA; 500 nM), and potassium (30 mM) were determined fromperifused islets cultured for 22-24 h in CMRL-1066 medium (controlcultured) or islets cultured in the additional presence of 500 nM TPA.Islet content of protein kinase C  (PKC) and serine and threoninephosphoprotein patterns were also monitored after the culture period.Compared with freshly isolated islets, culturing alone had no adverseeffect on the capacity of TPA or 30 mM potassium to stimulate secretionor on the islet content of PKC. In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKC by>95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPAslightly improved the insulin secretory response to 15 mM glucosecompared with control-cultured islets; however, sustained rates of 15 mM glucose-induced secretion from these islets were significantly lessthan the responses of freshly isolated islets. Islets cultured in TPAresponded to 30 mM potassium with a markedly amplified insulinsecretory response that was abolished by nitrendipine. Enhancedphosphorylation of several islet proteins was also observed inTPA-cultured islets compared with control-cultured islets. Thesefindings demonstrate that culturing alone impairs glucose-induced secretion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture medium. Sustained phosphorylation of several islet proteins inTPA-cultured islets may account, at least in part, for augmented calcium-dependent secretion.

     

Co-expression and regulation of connexins 36 and 43 in cultured neonatal rat pancreatic islets

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of beta cells observed at in vitro conditions.     

Beneficial effect of pretreatment of islets with fibronectin on glucose tolerance after islet transplantation.

The scarcity of available islets is an obstacle for clinically successful islet transplantation. One solution might be to increase the efficacy of the limited islets. Isolated islets are exposed to a variety of cellular stressors, and disruption of the cell-matrix connections damages islets. We examined the effect of fibronectin, a major component of the extracellular matrix, on islet viability, mass and function, and also examined whether fibronectin-treated islets improved the results of islet transplantation. Islets cultured with fibronectin for 48 hours maintained higher cell viability (0.146 +/- 0.010 vs. 0.173 +/- 0.007 by MTT assay), and also had a greater insulin and DNA content (86.8 +/- 3.6 vs. 72.8 +/- 3.2 ng/islet and 35.2 +/- 1.4 vs. 30.0 +/- 1.5 ng/islet, respectively) than islets cultured without fibronectin (control). Absolute values of insulin secretion were higher in fibronectin-treated islets than in controls; however, the ratio of stimulated insulin secretion to basal secretion was not significantly different (206.9 +/- 23.3 vs. 191.7 +/- 20.2% when the insulin response to 16.7 mmol/l glucose was compared to that of 3.3 mmol/l glucose); the higher insulin secretion was thus mainly due to larger islet cell mass. The rats transplanted with fibronectin-treated islets had lower plasma glucose and higher plasma insulin levels within 2 weeks after transplantation, and had more favorable glucose tolerance 9 weeks after transplantation. These results indicate that cultivation with fibronectin might preserve islet cell viability, mass and insulin secretory function, which could improve glucose tolerance following islet transplantation.     

Glucose regulates glucokinase activity in cultured islets from rat pancreas

In this study, we have used isolated pancreatic islets cultured for 7 days in 3 or 30 mM glucose to explore whether glucokinase is induced or activated by high glucose concentrations and has related enzyme activity to glucose-stimulated insulin release. Islets cultured in low glucose medium or low glucose medium plus 350 ng/ml insulin did not respond to high glucose stimulation. Islets cultured in medium containing high glucose concentrations showed a high rate of basal insulin secretion when perifused with 5 mM glucose, and the insulin release was greatly augmented in a biphasic secretion profile when the glucose concentration was raised to 16 mM. Islet glucokinase and hexokinase activities were determined by a sensitive and specific fluorometric method. Glucokinase activity was reduced to approximately 50% in islets cultured in low glucose medium with or without insulin present compared to results with fresh islets. However, islets cultured in 30 mM glucose showed that glucokinase activity was elevated to 236% compared to results with fresh islets. It is concluded that (a) glucose is the physiological regulator of glucokinase in the islet of Langerhans and that (b) the activity of glucokinase plays a crucial role in glucose-induced insulin secretion.     

INGAP-related pentadecapeptide: its modulatory effect upon insulin secretion

We examined the effects of a pentadecapeptide having the 104-118 aminoacid sequence of islet neogenesis-associated protein (INGAP-PP) on insulin secretion, and the morphological characteristics of adult and neonatal pancreatic rat islets cultured in RPMI and 10 mM glucose for 4 days, with or without different INGAP-PP concentrations (0.1-100 mug/ml). A scrambled 15 aminoacid peptide was used as control for the specificity of INGAP-PP effect. Cultured neonatal and adult islets released insulin in response to glucose (2.8-16.7 mM) in a dose-dependent manner, and to leucine and arginine (10 mM). In all cases, the response was greater in adult islets. INGAP-PP added to the culture medium significantly enhanced glucose- and aminoacid-induced insulin release in both adult and newborn rats; however, no changes were observed with the scrambled peptide. Similar results were obtained incubating freshly isolated adult rat islets with INGAP-PP. Whereas INGAP-PP did not induce significant changes in islet survival rate or proportion/number of islet cells, it increased significantly beta-cell size. This first demonstration of the enhancing effect of INGAP-PP on the beta-cell secretory response of adult and newborn islets opens a new avenue to study its production mechanism and potential use to increase the secretory capacity of endogenous islets in intact animals or of islets preserved for future transplants.     

Stimulation of 6-keto-prostaglandin F1 alpha release from isolated pancreatic islets by an insulinotropic concentration of glucose

In a glucose-free medium, the release of 6-keto-prostaglandin F1 alpha from isolated pancreatic islets was 1.67 pg/islet/60 min. The release was not altered by increasing the glucose concentration to 3.3 mM, whereas the release was significantly increased to 3.79 pg/islet/60 min by 16.7 mM glucose. Indomethacin (10 microM) and mepacrine (100 microM) markedly suppressed the 6-keto-prostaglandin F1 alpha release. The results indicate that the insulinotropic concentration of glucose enhances the enzymatic formation of 6-keto-prostaglandin F1 alpha in pancreatic islets. It seems highly possible that glucose enhances 6-keto-prostaglandin F1 alpha formation by stimulating phospholipase A2 in pancreatic islets.     

Alpha- and beta-anomeric preference of glucose-induced insulin secretion at physiological and higher glucose concentrations, respectively

We determined the anomeric preference of glucose phosphorylation by islet glucokinase, glucose utilization by pancreatic islets, and insulin secretion induced by glucose over a wide range of glucose concentrations. alpha-D-Glucose was phosphorylated faster than beta-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM). At 20 mM, there was no significant difference in phosphorylation rate between the two anomers. Similar patterns of anomeric preference were observed both in islet glucose utilization and in glucose-induced insulin secretion. The present study affords strong evidence that glucokinase is responsible for the anomeric preference of glucose-stimulated insulin secretion through anomeric discrimination in islet glucose utilization.     

Glucose-induced phospholipid-dependent protein phosphorylation in neonatal rat islets

The participation of calcium-activated, phospholipid-dependent protein kinase in the phosphorylation of endogenous islet proteins following the exposure of cultured, neonatal pancreatic islets to stimulatory glucose concentrations was investigated by two techniques. In the first technique, islets were prelabeled with 32Pi. The major endogenous substrates for glucose-induced phosphorylation had apparent molecular masses of 130,100 +/- 1010, 100,000 +/- 700, 80,400 +/- 890, 58,100 +/- 1200, 39,800 +/- 700, and 29,400 +/- 700 Da. In the presence of 12-O-tetradecanoylphorbol 13-acetate (2 microM), an activator of calcium-activated phospholipid-dependent kinase, there was enhanced phosphorylation of proteins of 80,000, 40,000, and 29,000 Da. In the second technique, exogenous phosphorylation by [gamma-32P]ATP of proteins in a postnuclear particulate fraction was studied in the presence and absence of cofactors for Ca2+-activated, phospholipid-dependent protein kinase (Ca2+, phosphatidylserine, and unsaturated diolein). These studies were performed in islets preexposed to low (1.7 mM) or high (16.7 mM) glucose concentration prior to preparation of the postnuclear particulate fraction. Following exposure of islets to low glucose concentration, three substrates (apparent molecular masses 40,500 +/- 600, 57,100 +/- 700, and 79,400 +/- 600 Da) in the postnuclear particulate fraction exhibited enhanced phosphorylation in the presence of calcium ions, phosphatidylserine, and unsaturated diolein. In preparations of islets preexposed to 16.7 mM glucose, the phosphorylation of the protein of molecular mass about 40,000 Da was significantly reduced, indicating prior phosphorylation of the acceptor sites on this substrate in response to glucose exposure. It is concluded that stimulation of neonatal cultured islets by glucose induces the acute changes in calcium ion, phospholipid, and diacylglycerol concentration required to activate the calcium-activated phospholipid-dependent protein kinase and that the islet postnuclear particulate fraction contains at least one specific substrate for this kinase.     

The role of old age in the effects of glucose on insulin secretion, pentosephosphate shunt activity, pyridine nucleotides and glutathione of rat pancreatic islets

In pancreatic islets of adult (three month) and old (24 month) rats the effect of glucose on glucose oxidation, pyridine nucleotides, glutathione and insulin secretion was studied. DNA content was similar in both groups of animals; however, islets of old rats exhibited 30% less insulin content. While glucose-induced (16.7 mM) insulin secretion in islets of old rats was approximately 50% less than in islets of adults, no significant difference was observed in the insulin releasing effect of theophylline (1 mM). Although islet production of 14CO2 in the presence of 16.7 mM glucose increased equally in both groups, elevation of glucose failed to increase the percentage of total glucose oxidation via the pentose phosphate shunt in islets of old rats. Elevation of glucose increased the NADPH/NADP and the NADH/NAD ratio in both groups of islets in a similar manner. The effect of glucose on the GSH/GSSG ratio revealed a dose-related increase in the islets of adult rats, whereas islets of old rats did not respond to elevation of glucose. Our data seem to indicate that the lower secretory response of islets of old rats is related to the failure of glucose to increase the GSH/GSSG ratio. In contrast the insulin release induced by theophylline does not appear to depend on islet thiols.     

3H-cyclosporine internalization and secretion by human fetal pancreatic islets

Human fetal pancreatic islets were isolated from 16- to 20-week-old fetuses by a collagenase technique and cultured 48 hr in RPMI 1640 containing 10% human adult serum and unlabeled 0 to 5 micrograms cyclosporine A (CsA)/ml. Insulin secretory capacity of human fetal islets was expressed as a fractional stimulatory ratio FSR = F2/F1 of the fractional secretion rates during two successive 1 hr static incubations first with 2 mM glucose (F1) to stabilize secretion followed by maximal stimulus, i.e., 25 mM glucose plus 10 mM L-leucine and 10 mM L-arginine (F2). Unlabeled CsA at the above concentrations had no significant effects on the insulin secretory capacity expressed by FSR-values. Studies of net uptake of 3H-CsA by islets cultured for varying periods up to 40 hr and expressed as picomole 3H-CsA per picomole islet insulin content demonstrated that uptake rate was slow and did not reach isotopic equilibrium over the 40 hr of culture. When isolated fetal islets were cultured for 48 hr in the presence of 3H-CsA and varying concentrations of unlabeled CsA it was found during two successive 1 hr static incubations that fetal islets secrete insulin concomitantly with 3H-CsA following maximal stimulus for secretion. An optimal secretory molar ratio of 3H-CsA to insulin of 4.0 +/- 1.3 (n = 7) was found after islets were cultured 48 hr in the presence of a saturating 2.128 micrograms 3H-CsA per milliliter culture medium. In three successive 30-min static incubations of 3H-CsA loaded islets, first with low glucose, followed by high glucose plus L-arginine and L-leucine, and finally with high glucose plus L-arginine and L-leucine and 10 mM theophylline, the proportional fractional secretion rates of insulin and 3H-CsA were of the same magnitude. It is concluded that human fetal pancreatic islets during 48 hr of culture in the presence of pharmacologically relevant concentrations of CsA can internalize the drug, which is compartmentalized and concomitantly secreted with insulin following maximal stimuli. Transplanted human fetal islets utilized as delivering units for CsA could be beneficial for the induction of immunotolerance to allografted fetal islets.     

SREBP1 is required for the induction by glucose of pancreatic beta-cell genes involved in glucose sensing

Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have reexamined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/l glucose led to enhanced secretion at both basal (3 mmol/l) and stimulatory (17 mmol/l) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K(+) channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/l glucose, SREBP1-deficient (SREBP1(-/-)) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1(-/-) mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.     

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.     

Intact pancreatic islets and dispersed beta-cells both generate intracellular calcium oscillations but differ in their responsiveness to glucose

Pancreatic islets produce pulses of insulin and other hormones that maintain normal glucose homeostasis. These micro-organs possess exquisite glucose-sensing capabilities, allowing for precise changes in pulsatile insulin secretion in response to small changes in glucose. When communication among these cells is disrupted, precision glucose sensing falters. We measured intracellular calcium patterns in 6-mM-steps between 0 and 16 mM glucose, and also more finely in 2-mM-steps from 8 to 12 mM glucose, to compare glucose sensing systematically among intact islets and dispersed islet cells derived from the same mouse pancreas in vitro. The calcium activity of intact islets was uniformly low (quiescent) below 4 mM glucose and active above 8 mM glucose, whereas dispersed beta-cells displayed a broader activation range (2-to-10 mM). Intact islets exhibited calcium oscillations with 2-to-5-min periods, yet beta-cells exhibited longer 7–10 min periods. In every case, intact islets showed changes in activity with each 6-mM-glucose step, whereas dispersed islet cells displayed a continuum of calcium responses ranging from islet-like patterns to stable oscillations unaffected by changes in glucose concentration. These differences were also observed for 2-mM-glucose steps. Despite the diversity of dispersed beta-cell responses to glucose, the sum of all activity produced a glucose dose-response curve that was surprisingly similar to the curve for intact islets, arguing against the importance of “hub cells” for function. Beta-cells thus retain many of the features of islets, but some are more islet-like than others. Determining the molecular underpinnings of these variations could be valuable for future studies of stem-cell-derived beta-cell therapies.