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.     

A functional analysis on isolated rat islets of Langerhans: effects of dimethylsulfoxide and low-temperature preservation

Cryopreservation of pancreatic islets of Langerhans offers the possibility of storage of sufficient quantities of this tissue for transplantation in the treatment of certain forms of diabetes, as well as providing a means of precise histocompatibility matching. In these studies, the effects of dimethylsulfoxide and various cooling rates on islet function are examined. These studies demonstrate that islets treated with 1.4 M dimethylsulfoxide and slowly cooled at a rate of 0.3 degrees C/min release insulin biphasically upon glucose challenge. In addition, this stimulated release is significantly improved (P less than 0.05) by increasing the duration of post-thaw culture. After thawing, these cryopreserved islets also retain the capacity to synthesize insulin. Islets frozen at faster cooling rates (3, 14, and 48 degrees C/min) exhibit varying degrees of glucose-induced insulin release, indicative of freeze-induced damage. These manifestations of freeze-induced damage include high basal (nonstimulatory) insulin release rates, little or no increase in the stimulated rate versus the nonstimulated rate, and failure of the stimulated release to return to basal levels when the glucose concentration is reduced.     

Effect of thawing rate and post-thaw culture on the cryopreserved fetal rat islets: functional and morphological correlation

The ability of the fetal pancreatic islet cells to multiply rendered them a potential tissue for transplantation studies to cure diabetes. A bank of fetal islets could be created with proper storage in liquid nitrogen. The aim of this study is to evaluate the effect of thawing rate and post-thaw culture on the structural and functional integrity of isolated cryopreserved islets of rat fetuses. Fetal rat islets were isolated by the collagenase digestion, cultured for three days, and then cryopreserved using dimethylsulphoxide as cryoprotectant and the step-rate cooling to -40 degrees C before immersing them in liquid nitrogen. The islets were thawed by the slow or fast warming rates using hyperosmolar sucrose solution and then cultured for 1 or 2 days. Insulin and C-peptide contents of the slow thawed islets were higher than those of the control. In the fast thawed islets the contents were similar to those of the control. Insulin and C-peptide release in response to glucose for the slow thawed islets were lower than those of the control and in the fast thawed islets they were similar to that of the control. Histological examination showed irregular periphery and fragmented central part of the large slowly thawed islets, which showed also variable immunohistochemical reaction to anti-insulin serum, ranging from strongly positive reaction to markedly weak reaction. Fast thawed islets showed mostly regular periphery and their reaction to the anti-insulin serum was slightly weaker than that of the control islets. It was concluded that fast thawing and post-thaw culture is much better than slow thawing, as indicated by nearly normal insulin and C-peptide content and release and intact structural integrity.     

Curcumin treatment enhances islet recovery by induction of heat shock response proteins, Hsp70 and heme oxygenase-1, during cryopreservation

Limited recovery of islets post-cryopreservation influences graft survival and transplantation efficiency during diabetes treatment. As curcumin, a potent antioxidant/radical scavenging compound, protects islets against beta cell toxins, we hypothesized that inclusion of curcumin during cryopreservation or during post-thaw culture or both may rescue islets from cryoinjury. To test the effect of curcumin inclusion on islet recovery murine islets were isolated by the collagenase digestion, cultured for 48 h, cryopreserved using dimethylsulphoxide as cryoprotectant -- with or without curcumin (10 microM) -- and then slow cooled to -40 degrees C before immersing them in liquid nitrogen for 7 days. Following rapid thawing with sucrose gradient and 24 h post-thaw culture -- in presence or absence of curcumin (10 microM) -- islet viability and functionality were determined. Islet recovery in curcumin treated groups was significantly higher than in groups where islets were cryopreserved without curcumin. Islets cryopreserved with curcumin also showed more intact islets as well as better morphology as compared to islets cryopreserved without curcumin. Curcumin treated islets also showed significant inhibition of ROS generation as compared to islets cryopreserved without curcumin. Glucose responsiveness and insulin secretion in islets cryopreserved with curcumin was equal to that of the freshly isolated islets as against islets cryopreserved without curcumin. Elevated level of Hsp 70 and HO-1 were observed in islets cryopreserved with curcumin and may contribute to curcumin-induced islet rescue. Hence, we conclude that inclusion of curcumin into cryopreservation medium inhibits ROS generation and corresponding islet damage and dysfunction.     

Viability tests of cryopreserved endocrine pancreatic cells

Collagenase-isolated islets, which had been cultured for 1 week, were frozen at two different cooling rates. Islets frozen at 5 degrees C/min behaved from a functional point of view very similarly to that of nonfrozen, cultured control islets, except for a reduced maximal insulin secretory capacity and a reduced insulin content. Slowly frozen islets (0.5 degrees C/min), however, displayed reduced rates of both proinsulin biosynthesis and glucose oxidation. It is concluded that isolated islets can be cryopreserved with great success and that the methods of choice for viability tests are those characterizing the dynamics of insulin secretory capacity of the cryopreserved islets.     

Lack of glucose-induced functional maturation during long-term culture of human fetal islet cells

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.     

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.     

Preservation of the effects of pregnancy on rat islets of Langerhans in tissue culture.

Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.     

The significance of culture for successful cryopreservation of isolated pancreatic islets of Langerhans

It was the aim of the present study to investigate the significance of culture before and after freeze-thawing of isolated mouse pancreatic islets. To evaluate the impact of culture before freezing (5 degrees C/min; 2 M dimethyl sulfoxide), islets were frozen either directly after isolation or after 2, 4, or 7 days of culture in medium RPMI 1640. The culture period after thawing was 7 days. Islets immediately frozen exhibited virtually no (pro)insulin biosynthesis and also a severe inhibition of glucose-stimulated insulin release. The precultured (2-7 days), frozen islets synthesized and released insulin at rates comparable to those of nonfrozen, cultured islets. Studies of the effects of culture after freeze-thawing were performed after a 3-day culture period prior to freezing. The (pro)insulin biosynthetic rates did not differ between islets cultured for 0-7 days after thawing. There was an apparent increase of glucose-stimulated insulin release when the islets were cultured for more than 2 days after thawing. It may be that the decreased viability of islets frozen immediately after isolation was due to minor cell damage induced by the collagenase incubation. During culture the islets may recover and become more resistant to freeze-damage. The beneficial effect of culture after thawing may reflect the loss of damaged cells, which otherwise would influence the results of the viability tests.     

Vitrification of mouse islets of Langerhans: comparison with a more conventional freezing method

The possibility of cryopreservation of islets of Langerhans by vitrification using a mixture of cryoprotectants was investigated and the results were compared with a more conventional freezing method using Me2SO as cryoprotectant. Isolated mouse islets were divided into three groups: (1) control islets cultured for 6 days, (2) islets which were cryopreserved by vitrification after 2 days of culture, and (3) islets frozen in 1.5 M Me2SO after 2 days of culture. After warming, islets from groups 2 and 3 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, survival during postwarming culture, morphology, and capability to reverse streptozotocin-induced diabetes. The insulin secretion in islets from all groups could be stimulated by a factor 5 or more by an increase in the concentration of glucose from 2.5 to 25 mM. The secretion of insulin in the presence of 2.5 mM glucose was similar in all groups of islets. The secretion of insulin in the presence of 25 mM glucose was slightly but not significantly lower in the cryopreserved islets than in the control noncryopreserved islets. The survival of islets during postwarming culture was comparable after cryopreservation with both methods, and islets from both groups could lower serum glucose in streptozotocin diabetic mice. We conclude that islets cryopreserved by the vitrification method are functional in vitro and in vivo. This method is quick, simple, and cheap because the use of complicated freezing equipment is avoided.     

Reduced sensitivity to dexamethasone of pancreatic islets from obese (fa/fa) rats.

The direct effects of dexamethasone exposure on insulin secretion from islets of fa/fa rats and their lean littermates (Fa/?) were compared. After 72 h culture in 1 nM dexamethasone, glucose (27.5 mM)-stimulated insulin secretion over 90 min from islets of lean rats was significantly decreased compared with islets cultured without dexamethasone (12.9 +/- 1.4 vs. 5.7 +/- 1.0% of total islet content, p < 0.05). Higher doses of dexamethasone for 24-48 h culture produced similar effects. For islets of fa/fa rats, the minimum inhibitory concentration of dexamethasone was 10-fold higher, and islets required at least 48 h exposure for inhibitory effects to be observed. Dexamethasone also decreased the insulin response by islets to glybenclamide, indicating that dexamethasone effects were not specific to glucose transport or metabolism. The results suggest that islets of fa/fa rats may be less sensitive to direct inhibitory effects of glucocorticoids on glucose-stimulated insulin release than islets of lean animals.     

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.     

Beta-cell function in isolated human pancreatic islets in long-term tissue culture

Human pancreatic islets were isolated by collagenase treatment of pancreatic tissue obtained from 27 individuals aged 12 to 69 years. The islets were maintained free floating in tissue culture medium RPMI 1640 supplemented with calf or human serum. In two cases the insulin production was followed up to nearly two years. The insulin production rate of the individual islet preparations varied between 0.2 and 8 ng per islet per day. No significant correlation with donor age or sex was found. The glucose concentration in the medium influenced the insulin release in a dose dependent manner. The acute response of the cultured islets to glucose was evaluated both by batch incubation and by perifusion. Both in the acute and the chronic experiments maximal insulin release was found at 10 mM glucose. In conclusion, these experiments indicate that viable islets of Langerhans can be obtained from adult human pancreatic tissue and that their beta-cell function can be maintained for up to two years. The variation in insulin production rate could not be ascribed to age or sex and may reflect both physiological and methodological factors.     

Successful cultivation of isolated islets of Langerhans without attachment: relationship between Glucose- and theophylline-induced insulin release and insulin content in rats islets after cultivation.

The effect of various inhibitors of insulin secretion such as mannoheptulose (20 mM), atropine (1 mM), diphenylhydantoin (20 microng/ml), high concentration of Mg++ (5.3 mM) in the presence of 20 mM glucose (control) on insulin content and secretion from collagenase-isolated rat pancreatic islets was studied in vitro by cultivation of islets up to 5 or 9 days in glass Petri dishes without attachment. In a following short-term incubation for 60 min the glucose-induced insulin release without and with theophylline (5 mM) was investigated. Islets cultivated at 5 mM glucose and at 20 mM glucose with the inhibitors mannoheptulose or atropine lost the responsiveness to glucose and theophylline whereas such islets cultivated at 20 mM glucose alone or with diphenylhydantoin (DPH) or 5.3 mg Mg++ showed a stimulation of insulin secretion by glucose and theophylline. Compared, however, with freshly isolated islets all cultivated islets were restricted in their maximal glucose response and this defect was not evoked alone by quantitative changes in islet insulin content. Nevertheless, culture conditions which facilitate a net increase of insulin (content and release) during cultivation influenced also positively the glucose-induced insulin release without and with 5 mM theophylline in the following short-term experiments.     

Islet transplantation in experimental diabetes of the rat. XIII. Cryopreservation reduces MHC class II but not class I antigens of rat pancreatic islets.

Pretreatment of islet allografts prior to transplantation may reduce islet immunogenicity and prolong graft acceptance. We have studied the MHC antigen reducing effect of cryopreservation onto rat pancreatic islets performing indirect immunofluorescence tests and peroxidase-anti-peroxidase staining (PAP). Three different freezing programs were used. Program A: 0.5 degrees C/min to -35 degrees C and 1 degree C/min from -35 to -100 degrees C. Program B: 2 degrees C/min to -35 degrees C and 6 degrees C/min from -35 to -100 degrees C. Program C: 0.25 degrees C/min to -40 degrees C. Cryopreservation clearly reduced the number of class II antigen positive cells per islet in all cases. Program A was most effective with 45.5% of class II antigen negative islets compared to 6.4% of class II antigen negative fresh islets as shown by indirect immunofluorescence. The class II antigen reducing effect of cryopreservation proved to be permanent and not only temporary. Reduced class II antigen expression of cryopreserved islets could not be reestablished by incubation of the islets with rat IFN. A combination of cryopreservation followed by a 10 day culture period proved to be most effective with 85.6% of class II antigen negative islets. In contrast, we could not show any effect of cryopreservation on class I antigen expression. Viability of the cryopreserved rat islets was shown in-vitro by glucose stimulated insulin secretion.     

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.     

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.     

Oxygen and temperature dependence of stimulated insulin secretion in isolated rat islets of Langerhans

The effects of lowered O2 tension on insulin secretion and changes in cellular energy parameters were investigated in isolated rat pancreatic islets perifused with buffers equilibrated with 21, 9, 5, and 1% oxygen and containing 5 mM glucose. Decreasing the external [O2] reduced the amount of insulin released in response to 16 mM glucose, 20 mM alpha-ketoisocaproic acid, and 40 mM KCl. Secretion elicited by high glucose or KCl had declined significantly at 9% oxygen, whereas that caused by alpha-ketoisocaproic acid became inhibited below 5% O2. Lowering the oxygen tension also decreased the ability of islets to respond with a rise in [ATP]/[ADP] upon stimulation with metabolic secretagogues. This reduction in the evoked increase in the nucleotide ratios paralleled the inhibition of stimulated insulin secretion. Addition of 2 mM amytal markedly decreased the islet energy level and eliminated the secretory response to 16 mM glucose. The results suggest that enhancement of B-cell energy production and a consequent rise in [ATP] (or [ATP]/[ADP]) are a necessary event for the hormone release elicited by high glucose and alpha-ketoisocaproic acid. A decrease in temperature inhibited insulin secretion with all three secretagogues tested. The energies of activation were similar for high glucose and KCl-induced secretion, about 20 kcal/mol, but were higher for alpha-ketoisocaproic acid, about 35 kcal/mol. At 28 degrees C, the [ATP]/[ADP] was larger than that at 38 degrees C (8 versus 5) and was not increased further upon addition of 16 mM glucose. It is suggested that a decrease in the rate of energy production at lowered temperatures may contribute to the inhibition of insulin release caused by metabolic secretagogues.     

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.     

Glucose- and time-dependence of islet amyloid formation in vitro

Islet amyloid contributes to the loss of beta-cell mass in type 2 diabetes. To examine the roles of glucose and time on amyloid formation, we developed a rapid in vitro model using isolated islets from human islet amyloid polypeptide (hIAPP) transgenic mice. Islets from hIAPP transgenic and non-transgenic mice were cultured for up to 7 days with either 5.5, 11.1, 16.7 or 33.3mmol/l glucose. At various time-points throughout the culture period, islets were harvested for determination of amyloid and beta-cell areas, and for measures of cell viability, insulin content, and secretion. Following culture of hIAPP transgenic islets in 16.7 or 33.3mmol/l glucose, amyloid formation was significantly increased compared to 5.5 or 11.1mmol/l glucose culture. Amyloid was detected as early as day 2 and increased in a time-dependent manner so that by day 7, a decrease in the proportion of beta-cell area in hIAPP transgenic islets was evident. When compared to non-transgenic islets after 7-day culture in 16.7mmol/l glucose, hIAPP transgenic islets were 24% less viable, had decreased beta-cell area and insulin content, but displayed no change in insulin secretion. Thus, we have developed a rapid in vitro model of light microscopy-visible islet amyloid formation that is both glucose- and time-dependent. Formation of amyloid in this model is associated with reduced cell viability and beta-cell loss but adequate functional adaptation. It thus enables studies investigating the mechanism(s) underlying the amyloid-associated loss of beta-cell mass in type 2 diabetes.