Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans

The possible involvement of chemiosmotic lysis of secretory granules in the exocytosis of insulin from pancreatic beta cells was investigated by comparing insulin release from isolated secretory granules, from intact islets of Langerhans, and from electrically permeabilised islets. Lysis of isolated granules was stimulated by ATP in the presence of Mg2+. ATP-induced granule lysis was pH and temperature dependent and was inhibited by collapsing the pH gradient across the granule membrane by removal of permeant anions, or by increasing the extragranular osmolarity. However, insulin secretion from intact islets in response to glucose, a phosphodiesterase inhibitor or a Ca2+ ionophore was only partially inhibited by anion replacement, while Ca2+ -induced insulin release from electrically permeabilised islets was not affected by altering the extragranular or intragranular pH. These results suggest that studies of the stability of isolated granules in vitro do not necessarily relate to insulin release from whole cells, and do not support a major role for chemiosmotic lysis of secretory granules in the exocytotic release of insulin.     

PCSK9 is expressed in pancreatic δ-cells and does not alter insulin secretion

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immunohistochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancreatic δ-cells, with no expression in α- and β-cells. PCSK9 seems not to be secreted by mouse isolated islets maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated islets, mainly in β-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin secretion in mouse islets. Finally, invivo glucose tolerance was similar in Pcsk9^+/+ and Pcsk9^−/− mice under basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that PCSK9 does not alter insulin secretion.     

Breakdown of exogenous insulin by langerhans islets of the pancreas in vitro

Pancreatic islets were isolated from Wistar rats, albino mice, spiny mice and sand rats (Psammomys obesus). Evidence is presented that pancreatic islets contain an enzyme system degrading insulin in the presence of glutathione or other sulfhydryl-containing compounds. Apparent K_m values for insulin and glutathione (in the presence of EDTA) are 14.0 μM (mol. wt 5700) and 1.28 mM, respectively. Maximum breakdown of ¹²⁵I-labeled insulin was found at about pH 7.2. After ultracentrifugation of islet homogenates the microsomal fraction contained the greatest relative specific insulin-degrading activity. The specific insulin-degrading actvitity was found to be higher in Wistar rats and albino mice than in spiny mice and sand rats. Starvation of Wistar rats for 72 h caused a decrease inthe enzymatic activity.     

Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

Glucose homeostasis is determined by insulin secretion from the ß-cells in pancreatic islets and by glucose uptake in skeletal muscle and other insulin target tissues. While glutamate dehydrogenase (GDH) senses mitochondrial energy supply and regulates insulin secretion, its role in the muscle has not been elucidated. Here we investigated the possible interplay between GDH and the cytosolic energy sensing enzyme 5′-AMP kinase (AMPK), in both isolated islets and myotubes from mice and humans. The green tea polyphenol epigallocatechin-3-gallate (EGCG) was used to inhibit GDH. Insulin secretion was reduced by EGCG upon glucose stimulation and blocked in response to glutamine combined with the allosteric GDH activator BCH (2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid). Insulin secretion was similarly decreased in islets of mice with ß-cell-targeted deletion of GDH (ßGlud1^−/−). EGCG did not further reduce insulin secretion in the mutant islets, validating its specificity. In human islets, EGCG attenuated both basal and nutrient-stimulated insulin secretion. Glutamine/BCH-induced lowering of AMPK phosphorylation did not operate in ßGlud1^−/− islets and was similarly prevented by EGCG in control islets, while high glucose systematically inactivated AMPK. In mouse C2C12 myotubes, like in islets, the inhibition of AMPK following GDH activation with glutamine/BCH was reversed by EGCG. Stimulation of GDH in primary human myotubes caused lowering of insulin-induced 2-deoxy-glucose uptake, partially counteracted by EGCG. Thus, mitochondrial energy provision through anaplerotic input via GDH influences the activity of the cytosolic energy sensor AMPK. EGCG may be useful in obesity by resensitizing insulin-resistant muscle while blunting hypersecretion of insulin in hypermetabolic states.     

Physicochemical characteristics of insulin secretion granules

beta-Granules were prepared from micro-dissected pancreatic islets of obese-hyperglycaemic mice. This fraction contained 60% of the insulin, 30% of the cytochrome oxidase, 16% of the acid phosphatase activity and 20% of the protein present in whole islets. The isolated granules retained a heavy metal during fractionation. Optimum conditions for granule stability were low ionic strength and pH6, the granules being unexpectedly fragile at pH7.4. The stability of the granules was unaffected by sucrose in the concentration range 50-320mm, but 1% (w/v) sodium deoxycholate released all insulin. A solubilizing effect was also noted with ATP and citrate. Spinning through 1.6m-sucrose yielded a further purification in relation to mitochondria and acid-phosphatase-carrying particles but virtually no purification in relation to protein. Electron microscopy revealed that the major contaminants were rough-surfaced vesicles and membranes. A separation of granules from acid phosphatase was achieved by phase distribution in polyethylene glycol and dextran. The location of the enzyme to the interphase was so pronounced in systems buffered with lithium phosphate that the technique may be used for future purification of acid-phosphatase-carrying particles from the beta-cells.     

Hyperplasia of Pancreatic Beta Cells and Improved Glucose Tolerance in Mice Deficient in the FXYD2 Subunit of Na,K-ATPase

Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2^−/− mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2^−/− mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2^−/− mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.     

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes ^1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass ^4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets ^4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function ^{5,6,8,9,16,17}.     

Delineation of glutamate pathways and secretory responses in pancreatic islets with β-cell–specific abrogation of the glutamate dehydrogenase

In pancreatic β-cells, glutamate dehydrogenase (GDH) modulates insulin secretion, although its function regarding specific secretagogues is unclear. This study investigated the role of GDH using a β-cell–specific GDH knockout mouse model, called βGlud1^−/−. The absence of GDH in islets isolated from βGlud1^–/– mice resulted in abrogation of insulin release evoked by glutamine combined with 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid or l-leucine. Reintroduction of GDH in βGlud1^–/– islets fully restored the secretory response. Regarding glucose stimulation, insulin secretion in islets isolated from βGlud1^–/– mice exhibited half of the response measured in control islets. The amplifying pathway, tested at stimulatory glucose concentrations in the presence of KCl and diazoxide, was markedly inhibited in βGlud1^–/– islets. On glucose stimulation, net synthesis of glutamate from α-ketoglutarate was impaired in GDH-deficient islets. Accordingly, glucose-induced elevation of glutamate levels observed in control islets was absent in βGlud1^–/– islets. Parallel biochemical pathways, namely alanine and aspartate aminotransferases, could not compensate for the lack of GDH. However, the secretory response to glucose was fully restored by the provision of cellular glutamate when βGlud1^–/– islets were exposed to dimethyl glutamate. This shows that permissive levels of glutamate are required for the full development of glucose-stimulated insulin secretion and that GDH plays an indispensable role in this process.     

Metabolic and secretory effects of methylamine in pancreatic islets

The metabolic and secretory effects of methylamine in rat pancreatic islets were investigated. Methylamine accumulated in islet cells, was incorporated into endogenous islet proteins, and inhibited the incorporation of [2,5-³H] histamine into either N,N-dimethylcasein or endogenous islet proteins. Methylamine (2 mM ) did not affect the oxidation of glucose or endogenous nutrients or the intracellular pH in islet cells. Glucose did not affect the activity of transglutaminase in islet homogenates, the uptake of ¹⁴C-methylamine by intact islets or its incorporation into endogenous islet proteins. Methylamine inhibited insulin release evoked by glucose, other nutrient secretagogues, and non-nutrient insulinotropic agents such as L -arginine or gliclazide. The inhibitory effect of methylamine upon insulin release was diminished in the presence of cytochalasin B or at low extracellular pH. Methylamine retarded the conversion of proinsulin to insulin. Trimethylamine (0.7 mM ) was more efficiently taken up by islet cells than methylamine (2.0 mM ), and yet caused only a modest inhibition of insulin release. These findings suggest that methylamine interferes with a late step in the secretory sequence, possibly by inhibiting the access of secretory granules to their exocytotic site.     

Cold-induced insulin release in vitro: Evidence for exocytosis

Summary Exposure of isolated pancreatic islets (mouse or rat) to low temperature (2° C) evoked a threefold increase in insulin release irrespective of the glucose concentration in the incubation medium. Cold-induced release was transient and rewarming to 37° C restored the sensitivity of B-cells to glucose stimulation. In islets cooled to 2° C, exocytotic profiles could easily be detected both by thin-section and freeze-fracture electron microscopy. As revealed by the freeze-fracture technique, the number of exocytotic profiles per membrane area was increased three-to fourfold as compared to islet cells incubated at 20° C. This was paralleled by intracellular fusion of secretory vesicles. Cold-induced insulin release was not affected by theophylline, cytochalasin B, omission of extracellular Ca⁺⁺ or D600. Replacement of extracellular Na⁺ with choline or sucrose suppressed the increase in insulin release and in frequency of exocytotic profiles recorded after exposure to 2° C. It is suggested that a redistribution of Ca⁺⁺ from intracellular stores, possibly mediated by an increase in intracellular Na⁺, triggers exocytosis of insulin granules upon exposure to cold.     

Slow oscillations persist in pancreatic beta cells lacking phosphofructokinase M

Pulsatile insulin secretion by pancreatic beta cells is necessary for tight glucose control in the body. Glycolytic oscillations have been proposed as the mechanism for generating the electrical oscillations underlying pulsatile insulin secretion. The glycolytic enzyme 6-phosphofructokinase-1 (PFK) synthesizes fructose-1,6-bisphosphate (FBP) from fructose-6-phosphate. It has been proposed that the slow electrical and Ca²⁺ oscillations (periods of 3–5 min) observed in islets result from allosteric feedback activation of PFKM by FBP. Pancreatic beta cells express three PFK isozymes: PFKL, PFKM, and PFKP. A prior study of mice that were engineered to lack PFKM using a gene-trap strategy to delete Pfkm produced a mosaic reduction in global Pfkm expression, but the islets isolated from the mice still exhibited slow Ca²⁺ oscillations. However, these islets still expressed residual PFKM protein. Thus, to more fully test the hypothesis that beta cell PFKM is responsible for slow islet oscillations, we made a beta-cell-specific knockout mouse that completely lacked PFKM. While PFKM deletion resulted in subtle metabolic changes in vivo, islets that were isolated from these mice continued to exhibit slow oscillations in electrical activity, beta cell Ca²⁺ concentrations, and glycolysis, as measured using PKAR, an FBP reporter/biosensor. Furthermore, simulations obtained with a mathematical model of beta cell activity shows that slow oscillations can persist despite PFKM loss provided that one of the other PFK isoforms, such as PFKP, is present, even if its level of expression is unchanged. Thus, while we believe that PFKM may be the main regulator of slow oscillations in wild-type islets, PFKP can provide functional redundancy. Our model also suggests that PFKM likely dominates, in vivo, because it outcompetes PFKP with its higher FBP affinity and lower ATP affinity. We thus propose that isoform redundancy may rescue key physiological processes of the beta cell in the absence of certain critical genes.     

Long lasting synchronization of calcium oscillations by cholinergic stimulation in isolated pancreatic islets

Individual mouse pancreatic islets exhibit oscillations in [Ca²⁺]_i and insulin secretion in response to glucose in vitro, but how the oscillations of a million islets are coordinated within the human pancreas in vivo is unclear. Islet to islet synchronization is necessary, however, for the pancreas to produce regular pulses of insulin. To determine whether neurohormone release within the pancreas might play a role in coordinating islet activity, [Ca²⁺]_i changes in 4-6 isolated mouse islets were simultaneously monitored before and after a transient pulse of a putative synchronizing agent. The degree of synchronicity was quantified using a novel analytical approach that yields a parameter that we call the “Synchronization Index”. Individual islets exhibited [Ca²⁺]_i oscillations with periods of 3-6 min, but were not synchronized under control conditions. However, raising islet [Ca²⁺]_i with a brief application of the cholinergic agonist carbachol (25 μM) or elevated KCl in glucose-containing saline rapidly synchronized islet [Ca²⁺]_i oscillations for ≥30 min, long after the synchronizing agent was removed. In contrast, the adrenergic agonists clonidine or norepinephrine, and the K_ATP channel inhibitor tolbutamide, failed to synchronize islets. Partial synchronization was observed, however, with the K_ATP channel opener diazoxide. The synchronizing action of carbachol depended on the glucose concentration used, suggesting that glucose metabolism was necessary for synchronization to occur. To understand how transiently perturbing islet [Ca²⁺]_i produced sustained synchronization, we used a mathematical model of islet oscillations in which complex oscillatory behavior results from the interaction between a fast electrical subsystem and a slower metabolic oscillator. Transient synchronization simulated by the model was mediated by resetting of the islet oscillators to a similar initial phase followed by transient “ringing” behavior, during which the model islets oscillated with a similar frequency. These results suggest that neurohormone release from intrapancreatic neurons could help synchronize islets in situ. Defects in this coordinating mechanism could contribute to the disrupted insulin secretion observed in Type 2 diabetes.     

Impaired Glucose Tolerance in a Mouse Model of Sidt2 Deficiency

Sidt2 was identified as a novel integral lysosomal membrane protein recently. We generated global Sidt2 knockout mice by gene targeting. These mice have a comparatively higher random and fasting glucose concentration. Intraperitoneal and oral glucose tolerance tests in Sidt2 knockout mice indicated glucose intolerance and decreased serum insulin level. Notably, the Sidt2^−/− mice had hypertrophic islets compared with control mice. By Western blot and immunofluorescence, Sidt2^−/− mouse islets were shown to have increased insulin protein, which actually contained more insulin secretory granules than their controls, demonstrated by electromicroscopy. Consistent with the in vivo study, isolated islet culture from the Sidt2^−/− mice produced less insulin when stimulated by a high concentration of glucose or a depolarizing concentration of KCl. Under electromicroscope less empty vesicles and more mature ones in Sidt2^−/− mice islets were observed, supporting impaired insulin secretory granule release. In conclusion, Sidt2 may play a critical role in the regulation of mouse insulin secretory granule secretion.     

Mechanism of Hyperinsulinism in Short-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Involves Activation of Glutamate Dehydrogenase

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh^−/−). The hadh^−/− mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh^−/− mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh^−/− mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh^−/− islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh^−/− islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh^−/− islets also have increased [U-¹⁴C]glutamine oxidation. In contrast, hadh^−/− mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh^−/− islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh^−/− islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.     

Synthesis of tritiated rat insulin with high specific activity: A method for radiolabeling the active site of insulin

Insulin was synthesized by rat islets from tritiated amino acids under conditions designed to achieve high specific activity. Islets were isolated by the collagenase method. Stores of unlabeled insulin were depleted by culturing them for 40 hours in the presence of 3-isobutyl-1-methylxanthine. The islets were then incubated for 22 hours in the presence of [³H]Isoleucine or [³H]Phenylalanine. These amino acids were chosen because they are specific markers from the A and B chains of insulin respectively. Labeled insulin was extracted from the islets and purified by gel filtration. Its biological activity was indistinguishable from monoiodinated insulin as assessed by binding to receptors on cultured human lymphocytes and by precipitation by anti-insulin antibodies. The specific activity was (18 Ci/mmole) and (37 Ci/mmole) for [³H]Ile and [³H]Phe insulin respectively.     

Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets

BACKGROUND INFORMATION: Pancreatic beta-cells require an optimal insulin content to allow instantaneous secretion of insulin. This is maintained by insulin biosynthesis and intracellular degradation of insulin. Degradation may be effected by crinophagy, i.e. the fusion of secretory granules with lysosomes. IL-1beta (interleukin 1beta) induces distinct changes of beta-cell lysosomes. To study the mechanisms for intracellular insulin degradation and crinophagy, isolated mouse pancreatic islets were exposed to IL-1beta and known pathways for IL-1beta actions were blocked. Intracellular insulin degradation was determined by following the fate of radioactively labelled insulin. Crinophagy was studied by ultrastructural analysis. The effects of blocking pathways for IL-1beta were monitored by measurements of nitrite and PGE(2) (prostaglandin E(2)). RESULTS: IL-1beta caused an enhancement of islet intracellular insulin degradation and an increase in the lysosomal incorporation of beta-cell secretory granules. The effects of IL-1beta were abolished by aminoguanidine, a selective inhibitor of inducible NOS (nitric oxide synthase), or by rofecoxib, a specific inhibitor of COX-2 (cyclo-oxygenase 2). In the absence of IL-1beta, nitroarginine, which is a selective inhibitor of constitutive NOS, caused a decrease in intracellular degradation of insulin in parallel with a decreased production of NO and PGE(2) by the islets. CONCLUSIONS: The correlation between the enhanced intracellular insulin degradation and lysosomal changes caused by IL-1beta suggests that insulin degradation may be effected by crinophagy. Under physiological conditions, significant beta-cell degradation of insulin may depend on the activity of COX-2, possibly stimulated by endogenous NO.     

Effect of Dexamethasone on Insulin Secretion: Examination of Underlying Mechanisms

ObjectiveTo study the mechanism of increased insulin secretion in response to short-term administration of dexamethasone.MethodsMale Wistar rats were injected intraperitoneally with dexamethasone (dexamethasone; 200 mcg/kg body weight per day) or saline for 3 consecutive days. Insulin secretion in response to glucose, ionomycin, and KCl was quantified in islets isolated from the animals, and the amount of glucokinase was measured by Western blot.ResultsDexamethasone-treated animals had 1.18-fold higher fasting blood glucose concentration and 6.5-fold increase in fasting serum insulin concentration compared with findings from animals injected with saline. Compared with islets isolated from control rats, islets from dexamethasone-treated rats secreted more insulin at 60 minutes in response to 5.5 mM glucose (416.4 vs 115.6 fmoles/10 islets, P = .011) and in response to 16.6 mM glucose (985.5 vs 520.6 fmoles/10 islets, P = .014); no change in insulin secretion was observed at 10 minutes. Insulin secretion from islets of dexamethasone-treated rats and control rats was not differentially augmented in response to either ionomycin or potassium chloride. Glucokinase expression was not altered by treatment with dexamethasone.ConclusionsAugmentation of insulin secretion in response to glucose in the pancreatic islets from dexamethasone-treated rats is preserved in islets studied in vitro. The increase in glucose-stimulated insulin secretion appears to be mediated by steps upstream to β -cell membrane depolarization and the attended increase in intracellular calcium in the signaling pathway of insulin secretion. (Endocr Pract. 2010;16:763-769)     

Interleukin-1 beta induces nitric oxide production and inhibits the activity of aconitase without decreasing glucose oxidation rates in isolated mouse pancreatic islets.

The aim of this investigation was to further characterize the process of interleukin-1 beta (IL-1 beta) induced nitric oxide production in isolated pancreatic islets. It was found that both IL-1 beta and nitroprusside increased islet nitrite production. This effect was paralleled by inhibition of islet aconitase activity and glucose oxidation rates. Neither trifluoroperazinen or aminopterin could prevent the IL-1 beta induced increase in nitrite production, aconitase inhibition and decrease in glucose oxidation rates. In a second series of experiments, isolated mouse pancreatic islets were exposed to IL-1 beta for 24 h and subsequently used for nitrite production, aconitase activity and glucose oxidation determinations. The islets responded to IL-1 beta with an increased nitrite production and a decreased activity of aconitase, whereas the islet glucose oxidation rates were not decreased. It is concluded that IL-1 beta in both rat and mouse islets induces nitric oxide formation and that this induction leads to the inhibition of the Krebs cycle enzyme aconitase. In rat islets this probably leads to an inhibited insulin secretion, whereas IL-1 beta in mouse islets suppresses insulin secretion by a non-mitochondrial mechanism.     

Development of In Vitro 3D TissueFlex? Islet Model for Diabetic Drug Efficacy Testing

Increasing individuals diagnosed with type II diabetes pose a strong demand for the development of more effective anti-diabetic drugs. However, expensive, ethically controversial animal-based screening for anti-diabetic compounds is not always predictive of the human response. The use of in vitro cell-based models in research presents obviously ethical and cost advantages over in vivo models. This study was to develop an in vitro three-dimensional (3D) perfused culture model of islets (Islet TF) for maintaining viability and functionality longer for diabetic drug efficacy tests. Briefly fresh isolated rat islets were encapsulated in ultrapure alginate and the encapsulated islets were cultured in TissueFlex^®, a multiple, parallel perfused microbioreactor system for 7 days. The encapsulated islets cultured statically in cell culture plates (3D static) and islets cultured in suspension (2D) were used as the comparisons. In this study we demonstrate for the first time that Islet TF model can maintain the in vitro islet viability, and more importantly, the elevated functionality in terms of insulin release and dynamic responses over a 7-day culture period. The Islet TF displays a high sensitivity in responding to drugs and drug dosages over conventional 2D and 3D static models. Actual drug administration in clinics could be simulated using the developed Islet TF model, and the patterns of insulin release response to the tested drugs were in agreement with the data obtained in vivo. Islet TF could be a more predictive in vitro model for routine short- and long-term anti-diabetic drug efficacy testing.