Dissociated insulin and islet amyloid polypeptide secretion from isolated rat pancreatic islets cocultured with human pancreatic adenocarcinoma cells

Abnormal insulin and islet amyloid polypeptide (IAPP) secretion are usually seen in patients with exocrine pancreatic cancer. The beta-cell dysfunction is a characteristic of the glucose intolerance found in pancreatic cancer patients. The effects of pancreatic cancer cells on insulin and IAPP secretion from beta cells are unclear. In this study, isolated rat pancreatic islets were cocultured with two human pancreatic adenocarcinoma cell lines (Panc-1 and HPAF) and a human colonic adenocarcinoma cell line (HT-29). As a control, islets were incubated in the absence of malignant cells. The accumulation of insulin and IAPP in culture media was measured by radioimmunoassay. Output of insulin and IAPP was decreased in islets cocultured with each malignant cell line. Molar ratio of secreted IAPP and insulin (IAPP/insulin) was increased in the islets cocultured with Panc-1 or HPAF cells, but not HT-29 cells. The decreased insulin and IAPP secretion were partly recovered after Panc-1, HPAF, or HT-29 cells were removed. The IAPP/insulin ratio was normalized after the removal of Panc-1 or HPAF cells. This study indicates that insulin and IAPP secretion are altered by the human adenocarcinoma cells investigated. The impairment induced by pancreatic adenocarcinoma cells is associated with a hypersecretion of IAPP relative to insulin on a molar basis.     

Intra- and extracellular amyloid fibrils are formed in cultured pancreatic islets of transgenic mice expressing human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is the constituent peptide of amyloid deposits found in the islets of non-insulin-dependent diabetic patients. Formation of islet amyloid is associated with a progressive destruction of insulin-producing beta cells. Factors responsible for the conversion of IAPP into insoluble amyloid fibrils are unknown. Both the amino acid sequence of human IAPP (hIAPP) and hypersecretion of hIAPP have been implicated as factors for amyloid fibril formation in man. We have generated transgenic mice using rat insulin promoter-hIAPP or rat IAPP (rIAPP) gene constructs. No fibrillar islet amyloid was detectable in vivo in these normoglycemic mice, although small amorphous perivascular accumulations of IAPP were observed in hIAPP mice only. To determine the effects of glucose on IAPP secretion and fibrillogenesis, pancreatic islets from transgenic and control mice were examined in vitro. Islet IAPP secretion and content were increased in transgenic islets compared with control islets. IAPP-immunoreactive fibrils were formed at both intra- and extracellular sites in isolated hIAPP islets cultured with glucose at 11.1 and 28 mM for only 7 days. At 28 mM glucose, fibrils were present in deep invaginations of beta cells as observed in non-insulin-dependent diabetic patients. No fibrils were present at low glucose concentrations in hIAPP islets or at any glucose concentration in rIAPP or control islets. Thus, glucose-induced expression and secretion of hIAPP in transgenic mouse islets can lead to formation of amyloid fibrils similar to that found in non-insulin-dependent diabetes mellitus.     

An in vitro model of early islet amyloid polypeptide (IAPP) fibrillogenesis using human IAPP-transgenic mouse islets.

The mechanisms underlying insufficient insulin secretion and loss of beta-cell mass in feline and human type 2 diabetes mellitus are incompletely understood. However, islet amyloid polypeptide (IAPP)-derived islet amyloidosis (IA) has been linked to increased rates of beta-cell apoptosis and, therefore, our goal was to develop an in vitro model of IAPP fibrillogenesis using isolated pancreatic islets from mice transgenic for human IAPP (hIAPP Tg mice). Islets from hIAPP Tg mice, from mice transgenic for non-amyloidogenic murine IAPP (mIAPP Tg mice), and from the FVB background strain were exposed to normal (5.5 mM) or high (28 mM) glucose conditions in cell culture for 8 days. On days 0 and 8, islets were collected for electron microscopy (EM). EM showed no abnormalities in the mIAPP Tg or FVB islets at either time point. On day 8, hIAPP Tg islets cultured at high glucose concentration formed extracellular IAPP-derived flocculent deposits. No significant differences in rates of apoptosis were found between groups. Our findings, therefore, show that in vitro culture of hIAPP Tg mouse islets under high glucose conditions produces a readily available and rapidly inducible model of IAPP-derived fibrillogenesis and enables the study of early phases of the molecular pathogenesis of IA.     

An in vitro model of early islet amyloid polypeptide (IAPP) fibrillogenesis using human IAPP-transgenic mouse islets

The mechanisms underlying insufficient insulin secretion and loss of β-cell mass in feline and human type 2 diabetes mellitus are incompletely understood. However, islet amyloid polypeptide (IAPP)-derived islet amyloidosis (IA) has been linked to increased rates of β-cell apoptosis and, therefore, our goal was to develop an in vitro model of IAPP fibrillogenesis using isolated pancreatic islets from mice transgenic for human IAPP (hIAPP Tg mice). Islets from hIAPP Tg mice, from mice transgenic for non-amyloidogenic murine IAPP (mIAPP Tg mice), and from the FVB background strain were exposed to normal (5.5 mM) or high (28 mM) glucose conditions in cell culture for 8 days. On days 0 and 8, islets were collected for electron microscopy (EM). EM showed no abnormalities in the mIAPP Tg or FVB islets at either time point. On day 8, hIAPP Tg islets cultured at high glucose concentration formed extracellular IAPP-derived flocculent deposits. No significant differences in rates of apoptosis were found between groups. Our findings, therefore, show that in vitro culture of hIAPP Tg mouse islets under high glucose conditions produces a readily available and rapidly inducible model of IAPP-derived fibrillogenesis and enables the study of early phases of the molecular pathogenesis of IA.     

Effects of islet amyloid polypeptide (IAPP) on insulin biosynthesis or secretion in rat islets and mouse beta TC3 cells. Biosynthesis of IAPP in mouse beta TC3 cells.

Effects of rat islet amyloid polypeptide (IAPP) on insulin biosynthesis and secretion were examined in isolated rat islets and mouse beta TC3 cells. Culture of islets or mouse beta TC3 cells for 24 h in the presence of 10(-6) M IAPP and 5.5 mM glucose had no effect on insulin mRNA levels. The rates of proinsulin biosynthesis were not altered in islets incubated in 10(-4)-10(-9) M IAPP. In beta TC3 cells, proinsulin biosynthesis was stimulated by glucose, though no effects of IAPP were shown. Addition of 10(-5) M IAPP to islets incubated in 11 mM glucose decreased the fractional insulin secretion rates; however, the secretion of insulin from beta TC3 cells was not affected by 10(-5) M IAPP. On the other hand, mouse beta TC3 cells expressed the elevated level of IAPP mRNA. Metabolic labeling of beta TC3 cells revealed the synthesis of both proIAPP and mature IAPP. In pulse chase experiments, proIAPP was processed to IAPP in a manner similar to proinsulin. These data indicate that IAPP is a possible polypeptide hormone synthesized in pancreatic beta cells though it is unlikely that IAPP is a physiologically relevant modulator of insulin biosynthesis or secretion.     

A role for glutathione peroxidase in protecting pancreatic beta cells against oxidative stress in a model of glucose toxicity

Antioxidant drugs have been reported to protect pancreatic islets from the adverse effects of chronic exposure to supraphysiological glucose concentrations. However, glucose has not been shown to increase intracellular oxidant load in islets, nor have the effects of increasing or inhibiting glutathione peroxidase (GPx) activity on islet resistance to sugar-induced oxidant stress been studied. We observed that high glucose concentrations increased intracellular peroxide levels in human islets and the pancreatic beta cell line, HIT-T15. Inhibition of gamma-glutamylcysteine synthetase (gammaGCS) by buthionine sulfoximine augmented the increase in islet peroxide and decrease in insulin mRNA levels, content, and secretion in islets and HIT-T15 cells induced by ribose. Adenoviral overexpression of GPx increased GPx activity and protected islets against adverse effects of ribose. These results demonstrate that glucose and ribose increase islet peroxide accumulation and that the adverse consequences of ribose-induced oxidative stress on insulin mRNA, content, and secretion can be augmented by a glutathione synthesis inhibitor and prevented by increasing islet GPx activity. These observations support the hypothesis that oxidative stress is one mechanism for glucose toxicity in pancreatic islets.     

Islet amyloid polypeptide in the islets of Langerhans: friend or foe?

Islet amyloid polypeptide (IAPP), or amylin, was originally discovered as the constituent peptide in amyloid occurring in human insulinomas and in pancreatic islets in human subjects with Type II (non-insulin-dependent) diabetes mellitus. Its normal expression in beta cells and its co-secretion with insulin in response to nutrient stimuli, suggest a metabolic function for the peptide. Specifically, IAPP has most frequently been shown to inhibit insulin secretion, implying that IAPP has a role in the regulation of islet hormone homeostasis. The physiological significance of IAPP in islets has been difficult to assess; very high IAPP concentrations are required to alter insulin secretion. Moreover, until recently, IAPP receptors have not been characterised at the molecular level, thus leaving the actual target cells for IAPP unidentified. Furthermore, in experimental diabetes in rodents, the ratio of IAPP expression to that of insulin invariably is increased. In view of the pleiotropic effects attributed to IAPP, such regulation could be both adverse and beneficial in diabetes. Metabolic characterisation of mice carrying a null mutation in the IAPP gene or which overexpress IAPP in beta cells have recently confirmed that IAPP is a physiological inhibitor of insulin secretion. Based on experiments in which IAPP-deficient mice develop a more severe form of alloxan-induced diabetes, we argue that the action of IAPP in the islets normally is beneficial for beta-cell function and survival; thus, the established up regulation of IAPP expression compared with that of insulin in experimental rodent diabetes could serve to protect islets under metabolically challenging circumstances. [Diabetologia (2000) 43: 687–695]     

Diazoxide attenuates glucose-induced defects in first-phase insulin release and pulsatile insulin secretion in human islets

Humans with type-2 diabetes mellitus (TTDM) have hyperglycemia ( approximately 11 mM) and impaired glucose-mediated insulin secretion characterized by impaired first-phase insulin release (FPIR) and pulsatile insulin release. Culture of islets from nondiabetic humans in very high glucose concentrations ( approximately 20-30 mM) for 96 h causes impaired FPIR. We sought to determine 1). whether human islets cultured at a glucose concentration of approximately 11 mM (comparable to TTDM) recapitulates impaired insulin secretion in TTDM, specifically impaired FPIR and insulin pulse mass with an increased proinsulin/insulin (PI/I) secretion ratio; and 2). whether these changes can be attenuated by addition of diazoxide to islets cultured with 11 mM glucose. Islets cultured with 11 mM glucose for 96 h had 75% depleted insulin stores (P < 0.05), decreased FPIR and insulin pulse mass (P < 0.05), and an approximately 3-fold increase in the ratio of PI/I islet content and in secretion ratio (P < 0.05). Addition of diazoxide to islets cultured with 11 mM glucose decreased insulin secretion during static incubation, leading to relative preservation of insulin stores and enhanced insulin secretion during subsequent perifusion; FPIR increased by 162% (P < 0.05) and insulin pulse mass by 150% (P < 0.05) vs. no diazoxide. The mean islet PI/I content and islet PI/I secretion ratio were also decreased by approximately 70% (P < 0.05) by prior addition of diazoxide to islets during culture with 11 mM glucose. FPIR and insulin pulse mass were related to islet insulin stores (P < 0.001 for FPIR and P < 0.001 for pulse amplitude). In conclusion, the pattern of defects of insulin secretion present in TTDM (impaired FPIR and pulsatile insulin secretion, increased PI/I ratio) can be recapitulated in human islets cultured with 11 mM glucose for 96 h. These defects can be at least partially offset by concurrent inhibition of insulin secretion by diazoxide, which also preserves insulin stores. Defective insulin secretion in TTDM may be, at least in part, due to depletion of available insulin stores secondary to chronic increased demand (insulin resistance and hyperglycemia) in the setting of a decreased beta-cell mass.     

Glucose affects in vitro maturation of fetal rat islets

Fetal pancreatic islets (21.5 days old) were cultured in RPMI 1640 containing either 2.8 or 11.1 mM glucose for 7 days. After the 7-day culture period, islets cultured in 2.8 mM glucose demonstrated a minimal first phase of insulin secretion in response to acute glucose stimulation, whereas islets cultured in 11.1 mM glucose demonstrated a biphasic insulin secretory pattern. Islets cultured in 11.1 mM glucose initiated insulin secretion at 4.4 +/- 0.1 mM glucose and plateaued at 11.6 mM glucose when exposed to a linear gradient. In addition, culture in 11.1 mM glucose increased DNA content (P less than 0.01) and [3H]thymidine incorporation (P less than 0.05) in fetal islets. However, ultrastructural morphometric analysis indicated that the actual number of beta-cells within islets cultured in either 2.8 or 11.1 mM glucose did not increase. The insulin contents of islets cultured in 2.8 and 11.1 mM glucose were 0.46 +/- 0.06 and 1.14 +/- 0.10 mU/islet, respectively. During subsequent glucose stimulation, islets cultured in 2.8 and 11.1 mM glucose released 3% and 5.6% of their total insulin content, respectively. Ultrastructural morphometric analysis indicated that 11.1 mM glucose stimulated an increase in the volume of individual beta-cells, i.e. hypertrophy. The hypertrophy of beta-cells within islets cultured in 11.1 mM glucose resulted in a concomitant increase in islet volume. Finally, the hypertrophy of beta-cells within islets cultured in 11.1 mM glucose was a result of increased volumes of mitochondria, secretory granules, and, to the greatest extent, endoplasmic reticulum. These findings indicate that glucose is a potent factor in the maturation of cultured fetal rat islets.     

Prolonged exposure of pancreatic beta cells to raised glucose concentrations results in increased cellular content of islet amyloid polypeptide precursors

Summary Most non-insulin dependent diabetic patients have amyloid deposits in their pancreatic islets. It is not known whether chronic hyperglycaemia contributes to the formation of amyloid fibrils from the islet amyloid polypeptide that is produced by the pancreatic beta cells. Since islet amyloid exhibits islet amyloid polypeptide precursors immunoreactivity, we examined whether sustained in vitro exposure to raised glucose increases the abundance of these precursors in human beta cells. After 6 days stimulation with 20 mmol/l glucose the cellular content of insulin but not islet amyloid polypeptide was decreased leading to an increase in the ratio of the latter over insulin (3.0 ± 0.6 vs 1.8 ± 0.3 after 6 mmol/l glucose culture, p < 0.05). Similar changes occurred in rat beta cells cultured for 3 days in the presence of 20 mmol/1 glucose plus 3-isobutyl-1-methylxanthine. Western blot analysis of cellular islet amyloid polypeptide after prolonged exposure to high glucose indicated the presence of higher proportions of its precursor- and intermediate forms. In human beta cells cultured in 20 mmol/l glucose, the major form corresponds to an intermediate species which exhibits an immunoreactivity for the N-flanking peptide, as is also the case in islet amyloid. We concluded that prolonged in vitro exposure of beta cells to raised glucose concentrations increases the relative proportion of islet amyloid polypeptide over insulin, as well as of its precursors over the mature form of islet amyloid polypeptide. [Diabetologia (1999) 42: 188–194] Received: 1 April 1998 and in final revised form: 13 October 1998     

Islet amyloid polypeptide regulates multiple steps in stimulus-secretion coupling of beta cells in rat pancreatic islets

Islet amyloid polypeptide (IAPP) is produced in pancreatic beta cells. Intraislet function of IAPP is still uncertain. In the present study, we investigated effects of IAPP and somatostatin on stimulus-secretion coupling of beta cells in isolated rat pancreatic islets. Insulin secretion induced by 22.2 mM glucose was increased by an IAPP antiserum (0.1%) or an IAPP antagonist (IAPP8-37, 10 microM). Pretreatment of islets with pertussis toxin (PTX) abolished the stimulating effect of IAPP8-37 on glucose-induced insulin secretion. In contrast, IAPP antiserum and IAPP8-37 did not change insulin secretion induced by 30 mM KCl. Somatostatin (1 nM) inhibited insulin secretion induced by 22.2 mM glucose, 10 mM L-arginine, 25 microM forskolin, and 200 microM carbachol. IAPP (10 microM) enhanced the inhibitory effect of somatostatin on insulin secretion induced by L-arginine or forskolin. PTX pretreatment abolished the effects of somatostatin and IAPP on arginine-induced insulin secretion. In conclusion, IAPP regulates multiple steps in signal transductions of beta cells. The effects of IAPP on beta cells are mediated by PTX-sensitive regulatory G proteins.     

Sustained exposure of toxically damaged mouse pancreatic islets to high glucose does not increase beta-cell dysfunction

The aim of this study was to clarify whether prolonged in-vitro exposure of either normal or damaged beta cells to a high glucose environment can be toxic to these cells. For this purpose NMRI mice were injected intravenously with a diabetogenic dose of streptozotocin (SZ; 160 mg/kg) or vehicle alone (controls). Their islets were isolated 15 min after the injection and subsequently maintained in culture for 21 days in the presence of 11.1 or 28 mmol glucose/l. After this period, during acute glucose stimulation, the control islets showed a marked increase in their insulin release in response to a high glucose stimulus. In the SZ-exposed islets there was a decrease in DNA and insulin contents, and a deficient insulin secretory response to glucose. However, in the SZ-damaged islets as well as in the control islets, culture with 28 mmol glucose/l compared with 11.1 mmol glucose/l did not impair islet retrieval after culture, islet DNA content or glucose-induced insulin release. Thus, the degree of damage was similar in the SZ-treated islets cultured at the two concentrations of glucose. These results suggest that glucose is not toxic to normal or damaged mouse pancreatic islets over a prolonged period in tissue culture.     

Inhibition of insulin secretion,but normal peripheral insulin sensitivity,in a patient with a malignant endocrine pancreatic tumour producing high amounts of an islet amyloid polypeptide-like molecule

Summary Islet amyloid polypeptide or amylin is a polypeptide secreted mainly from the pancreatic beta cells together with insulin upon stimulation. High levels of islet amyloid polypeptide have also been shown to increase the peripheral insulin resistance and consequently a role for islet amyloid polypeptide in the glucose homeostasis has been suggested. We have studied the glucose homeostasis in a patient with a malignant endocrine pancreatic tumour producing large amounts of an islet amyloid polypeptide-like molecule (about 400 times the upper reference level for islet amyloid polypeptide). This patient developed insulin-requiring diabetes mellitus shortly after the tumour diagnosis. Both intravenous and oral glucose tolerance tests revealed inhibited early responses in insulin and C-peptide release, but the insulin and C-peptide response to glucagon stimulation was less affected. Aneuglycaemic insulin clamp showed normal insulin-mediated glucose disposal. In vitro experiments, where isolated rat pancreatic islets were cultured with serum from the patient, showed a moderately decreased islet glucose oxidation rate and glucose-stimulated insulin release compared to islets cultured with serum from healthy subjects. However, culture of rat islets with normal human serum supplemented with synthetic rat islet amyloid polypeptide did not affect the glucose-stimulated insulin release. In conclusion, the observed effects show that the diabetic state in this patient was associated with an impaired glucose-stimulated insulin release but not with an increased peripheral insulin resistance. Thus, the results suggest that if islet amyloid polypeptide has diabetogenic effects they are more likely to be exerted at the level of insulin secretion than at the level of peripheral insulin sensitivity.     

Globular adiponectin augments insulin secretion from pancreatic islet beta cells at high glucose concentrations

Adiponectin plays an important role in improving insulin resistance and preventing atherosclerosis. However it has been rarely reported that adiponectin influences insulin secretion because its receptor was identified in human islet β cells. In order to investigate the direct effect of adiponectin on pancreatic islet β cells, we performed an insulin secretion test in purified rat islets, which were incubated with adiponectin (100 ng/mL) at low (3.3 mM) and high (16.7 mM) glucose concentrations. Furthermore, cell lysates were extracted from the adiponectin-treated islets for p-AMPKα assay. RTPCR and immunohistochemical examination showed both adiponectin receptor 1 (AdipoR1) and receptor 2 (A dipoR2) were expressed in islet cells and AdipoR1 was predominantly expressed. Insulin secretion was significantly increased in the presence of adiponectin for 6 h at high glucose concentration. Meanwhile, the levels of phosphorylated AMPK increased with adiponectin treatment at high glucose concentrations. It is concluded that adiponectin augments insulin secretion from pancreatic islet β cells at high glucose concentration through AMPK activation.     

Long term inhibitory effects of pancreastatin and diazepam binding inhibitor on pancreatic beta-cell deoxyribonucleic acid replication, polyamine content, and insulin secretion

The two peptides pancreastatin and diazepam binding inhibitor (DBI) were recently demonstrated in pancreatic islets and were shown to inhibit insulin secretion in short term experiments. In the present study we investigated long term effects of pancreastatin and DBI on the DNA synthesis, polyamine content, and insulin secretion of pancreatic beta-cells in tissue culture. For this purpose fetal rat pancreatic islets enriched in beta-cells were isolated and cultured for 3 days at different concentrations of rat pancreastatin and porcine DBI. It was found that pancreastatin dose-dependently decreased beta-cell DNA synthesis, reaching maximal inhibition at 100 nM. In parallel with this, pancreastatin also decreased insulin secretion and the islet contents of insulin and the polyamines spermidine and spermine. These effects were abolished by a high glucose concentration or addition of GH. Also, DBI evoked a dose-dependent inhibition of beta-cell DNA synthesis but affected neither the islet contents of insulin or polyamines nor insulin secretion. Like pancreastatin, DBI was ineffective in preventing the increased beta-cell DNA synthesis, insulin content, or secretion in response to high glucose or GH. It is concluded that pancreastatin and DBI inhibit beta-cell DNA synthesis and function in vitro. In the case of pancreastatin these inhibitory effects may be mediated by a decrease in islet polyamine content. It is suggested that pancreastatin and DBI may influence beta-cell replication and function in vivo in an autocrine or paracrine fashion.     

Biotin regulation of pancreatic glucokinase and insulin in primary cultured rat islets and in biotin-deficient rats.

Biotin has been reported to affect glucose homeostasis; however, its role on pancreatic islets of Langerhans has not been assessed. In this report, we demonstrate that physiologic concentrations of biotin stimulate glucokinase activity in rat islets in culture. Using the branched DNA (bDNA) assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of 41.5 +/- 13% and 81.3 +/- 19% at 12 and 24 h respectively in islets treated with [10(-6) M] biotin. Because glucokinase activity controls insulin secretion, we also investigated the effect of biotin on insulin release. Treatment with [10(-6) M] biotin for 24 h increased insulin secretion. We extended our studies by analyzing the effect of biotin deficiency on pancreatic islet glucokinase expression and activity, as well as insulin secretion. Our results show that islet glucokinase activity and mRNA are reduced by 50% in the biotin deficient rat. Insulin secretion in response to glucose was also impaired in islets isolated from the deficient rat. These data show that biotin affects pancreatic islet glucokinase activity and expression and insulin secretion in cultured islets.     

Survival of an islet β-cell in type-2 diabetes: curbing the effects of amyloid cytotoxicity

Type 2 diabetes mellitus is a chronic hyperglycaemic disorder caused by defective action and secretion of insulin. It is characterized by a progressive decline in pancreatic β-cell function and mass and the occurrence of insoluble amyloid deposits within the islets of Langerhans. These amyloid deposits comprise predominantly of fibrillar aggregates of the 37-amino acid human amylin (hA) monomer, also known as islet amyloid polypeptide (IAPP), which is co-secreted with insulin from pancreatic islet β-cells via the regulated secretory pathway. hA has a propensity to aggregate in vitro into fibrillar structures through the self-association of monomers that is largely mediated by an amyloidogenic region spanning amino acids 20-29 (reviewed in ref. 8).     

Dynamic perifusion to maintain and assess isolated pancreatic islets

Advances in human islet transplant techniques are hampered by the inability to assess the quality of isolated islets. A flow culture system was developed to perifuse isolated pancreatic islets or cultured beta-cell lines in order to continuously and noninvasively assess cell function and viability with high kinetic resolution. Continuous perifusion of large amounts of islet tissue as isolated from human pancreata enables the use of noninvasive measurement technologies not previously applied to islets. To compare dynamic perifusion of tissue at high density with conventional static cultures, we measured glucose-stimulated insulin secretion and O2 consumption of large amounts of INS-1 cells (45-65 x 10(6)) to confirm that perifused cells were adequately supplied with oxygen and nutrients and remained functionally responsive. Isolated human and monkey islets that were perifused for 18 h showed robust biphasic insulin secretion in response to a step increase in glucose, demonstrating the ability to maintain islets and the high kinetic resolution of the system. As an example of the system's ability to resolve multiple indicator dilution experiments, the retention of [3H]-glibenclamide was kinetically distinguished from that of an extracellular marker. In summary, the perifusion system is able to maintain healthy cells, assess insulin secretion and metabolite fluxes such as oxygen consumption and lactate production, and characterize the kinetics of the interaction between radiopharmaceuticals and islet cells. The ability to systematically assess the metabolic and functional viability of islets will facilitate the optimization of islet isolation procedures, islet transplantation studies, and islet storage methodologies.     

Effects of prolonged in vitro exposure to sulphonylureas on the function and survival of human islets

The direct effects of prolonged exposure to sulphonylureas on the function and survival of human islets are unknown. This study assessed the insulin content, glucose-stimulated insulin release, islet cell apoptosis, and mRNA expression of insulin and GLUT-1 in isolated human islets cultured in the presence of therapeutical concentrations of glimepiride (10 microM), glibenclamide (10 microM), or chlorpropamide (600 microM). Islets were prepared by collagenase digestion and density gradient purification from 18 multiorgan donors and were then exposed for 24 h to the different sulphonylureas. Insulin content decreased significantly following culture with any sulphonylurea compound. In response to an acute challenge with 3.3 and 16.7 mM glucose, insulin release from the control islets accounted for 1.9 +/- 0.5% and 4.9 +/- 1.7% of total insulin content (P<.01), respectively. Glucose responsiveness was preserved in islets precultured in the presence of glimepiride, whereas high glucose level did not elicit any significant increase of insulin secretion from islets preincubated with glibenclamide or chlorpropamide. These alterations were reverted by an additional 48-h incubation in drug-free conditions. The amount of apoptotic cells did not differ significantly among the experimental groups. Quantitative RT-PCR studies showed that, compared with the control islets, cells preincubated with glibenclamide or chlorpropamide had an increased expression of insulin mRNA, with no change in the expression of GLUT-1. In conclusion, prolonged exposure of human islets to different sulphonylureas causes different disturbances of islet cell function, with glimepiride showing milder effects, as compared with chlorpropamide and glibenclamide.     

Islet amyloid polypeptide (IAPP) secretion from islet cells and its plasma concentration in patients with non-insulin-dependent diabetes mellitus.

Islet amyloid polypeptide (IAPP/Amylin) is a novel peptide which was extracted from islet amyloid deposits in patients with non-insulin-dependent diabetes mellitus (NIDDM). However, its pattern of secretions and plasma concentrations under various conditions has not yet been made clear enough. In this study, we examined IAPP secretion from islet beta-cells in vitro using cultured islet cells of neonatal rat pancreas and plasma IAPP responses under various conditions in vivo in normal control subjects and patients with glucose intolerance. Our data revealed that (1) IAPP is co-secreted with insulin from islet cells of the rat pancreas by glucose and non-glucose stimuli; (2) fasting plasma IAPP levels in normal control subjects are 24.9 +/- 2.0 pg/ml and the molar ratio of IAPP/insulin is approximately 1/7; (3) fasting IAPP levels are high in obese patients and low in insulin-dependent diabetic patients, and the molar ratio of IAPP/C-peptide in NIDDM patients is lower than that in normal control subjects, suggesting the basal hyposecretion of IAPP relative to insulin in NIDDM; and (4) the obese patients who had a hyperresponsiveness of insulin relative to C-peptide had the hyperresponsiveness of IAPP relative to C-peptide during an oral glucose load, suggesting that IAPP may have some physiological effect in glucose metabolism.