The atypical antipsychotic clozapine impairs insulin secretion by inhibiting glucose metabolism and distal steps in rat pancreatic islets

Aims/hypothesis Diabetogenic effects of some atypical antipsychotic drugs have been reported, although the mechanisms are not fully understood. We investigated the long-term effects of culturing isolated rat pancreatic islets with atypical antipsychotic clozapine.Methods Glucose- and non-glucose-stimulated insulin secretion, glucose metabolism and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured in islets cultured with or without clozapine.Results Although acute incubation or 3-day culture with clozapine did not affect glucose-stimulated insulin secretion, clozapine suppressed glucose-stimulated insulin secretion by 53.2% at 1.0 μmol/l (therapeutic concentration) after 7 days of culture. Islet glucose oxidation and [Ca²⁺]_i elevation by high glucose were not affected after 3 days of culture, but clozapine significantly inhibited islet glucose oxidation, ATP production, and [Ca²⁺]_i elevation by high glucose after 7 days of culture. Moreover, 7 days of culture with clozapine inhibited insulin secretion stimulated by: (1) membrane depolarisation induced by high K⁺; (2) protein kinase C activation; and (3) mastoparan at 16.7 mmol/l glucose under stringent Ca²⁺-free conditions. Elevation of [Ca²⁺]_i by high K⁺-induced membrane depolarisation was similar in control and clozapine-treated islets. Clozapine, a muscarinic blocker, acutely inhibited carbachol-induced insulin secretion, as did atropine, whereas after 7 days of culture atropine did not have the inhibitory effect shown by clozapine after 7 days. The impairment of glucose-stimulated insulin secretion recovered 3 days after the removal of clozapine treatment.Conclusions/interpretation The present study demonstrated that the atypical antipsychotic drug clozapine directly impaired insulin secretion via multiple sites including glucose metabolism and the distal step in insulin exocytosis in a long-term culture condition. These mechanisms may be involved in the form of diabetes mellitus associated with atypical antipsychotic drugs.     

Can desensitization of the B-cell to D-glucose be simulated in cultured pancreatic islets?

Summary In order to investigate the phenomenon of B-cell desensitization to D-glucose, rat pancreatic islets were cultured for 20–44h in the presence of increasing concentrations of D-glucose in the 5.6 to 27.8 mM range, and then incubated for 30 to 120 min for measurement of secretory, metabolic and ionic variables. After culture in the presence of 5.6 mM D-glucose, the release of insulin evoked by D-glucose (16.7 mM) was less marked than that seen in islets cultured in the presence of 11.1 mM D-glucose. In the latter islets, the secretory response to D-glucose (8.3 mM or more) was still modest, especially over short periods of incubation, but was markedly enhanced by either theophylline or forskolin. The release of insulin evoked by D-glucose in the presence of theophylline was little affected by either Ca²⁺ concentration of the culture medium or length of culture period (20hvs 44h). The culture-induced alteration in the responsiveness to D-glucose coincided with a smaller relative increase of D-[5³H]glucose utilization, D-[U-¹⁴C]glucose oxidation or net⁴⁵Ca uptake at increasing concentrations of the hexose. It contrasted with a well-preserved secretory response to nonnutrient secretagogues. Although these findings could be interpreted as evidence of B-cell desensitization to D-glucose, the fact that the secretory behavior of the islets was not vastly different whether they were first cultured at physiological (8.3 mM) or higher (11.1 to 27.8 mM) concentrations of D-glucose suggests that this experimental design may not be an optimal model for the functional alteration of the B-cell in hyperglycemic non-insulin-dependent diabetic subjects.     

Effect of extracellular pH on insulin secretion and glucose metabolism in neonatal and adult rat pancreatic islets

Changes in extracellular pH are known to affect glucose-stimulated insulin secretion. In the present study, glucose metabolism in pancreatic islets cultured at different pHs was investigated. Also, for islet transplantation purposes, insulin secretion and glucose metabolism were compared in neonatal and adult islets at different pHs to determine which islet preparation is more tolerant to acidity and alkalinity. The results revealed a dependency of insulin secretion on the external pH in both neonatal and adult islets. Reduction of insulin secretion was observed at both the acidic and alkaline sides of pH 7.3. Glucose stimulated increases of insulin secretion in all cases. Similar results were obtained for ATP and pyruvate contents. Intracellular insulin increased with the increase of pH value. In contrast, calcium content decreased with the increase of pH. The results demonstrate that neonatal islets are more acid tolerant than adult islets. Both basal and glucose-stimulated insulin secretions, as well as other parameters of neonatal islets were significantly higher than those of adult islets in response to low pH. The differences under alkaline conditions were not significant but give an indication that neonatal islets are more tolerant to alkalinity than are adult islets. Received: 10 February 2001 / Accepted in revised form: 29 June 2001     

胰岛细胞系研究进展

由于细胞分化不完全及表型不稳定,目前仍没有非常适合代替原代胰岛β细胞进行功能研究用的细胞系。已经建立的胰岛细胞系主要分两类:动物胰岛β细胞瘤起源的细胞系(如RIN、INS细胞系)和人β细胞起源的细胞系,各种细胞系的生物学特征方面各不相同,但都保留了某些正常胰岛细胞功能特征。采用基因工程的方法和挑选更优越的亚系是目前改善细胞系功能的主要手段。     

Streptozotocin-induced FAD-glycerophosphate dehydrogenase suppression in pancreatic islets

In vitro, streptozotocin (1.0–2.0 mM) fails to exert any immediate effect on the activity of FAD-glycerophosphate dehydrogenase in either pancreatic islet homogenate or freshly isolated intact islets. However, when injected in vivo, streptozotocin (40 mg/kg body weight) lowers the specific activity of the FAD-linked enzyme in islet homogenates within 24 h, whilst causing little change in 2-ketoglutarate dehydrogenase and increasing glutamate dehydrogenase islet activity. In animals which became frankly hyperglycaemic as the result of the injection of streptozotocin, the activity of islet FAD-glycerophosphate dehydrogenase, measured 2 weeks after administration of the B-cell cytotoxic agent, was decreased to 10–20% of its control value. Neither insulin treatment nor riboflavin supplementation affected this enzymic defect. Even when the animals injected with streptozotocin remained virtually euglycaemic, the activity of islet FAD-glycerophosphate dehydrogenase was markedly decreased. This coincided with a preferential impairment of aerobic glycolysis, as judged from the ratio betweend-[3,4-¹⁴C]glucose oxidation andd-[5-³H] glucose utilization by the islets. It is proposed, therefore, that the administration of sub-diabetogenic amounts of streptozotocin to adult rats represents an alternative and easier approach to the study of B-cell dysfunction in this model of type 2 (non-insulin-dependent) diabetes than does streptozotocin injection in neonatal rats.     

Fate of3H- and14C-labelled A-4166 in pancreatic islets

The fate of³H- and¹⁴C-labelled A-4166 was examined in rat pancreatic islets. The net uptake of the meglitinide analogue by islets incubated for 60 min in the presence of 0.1 mM A-4166 and then submitted to repeated washes was close to 0.1 pmol/islet. It was significantly increased when the concentration ofd-glucose in the incubation medium was raised from 2.8 to 16.7 mM. No sizeable internalization of tritiated A-4166 into insulin-producing cells could be detected by autoradiography. These findings suggest that the interaction of A-4166 with the beta-cell may be restricted to its insertion on the plasma membrane and binding to sulphonylurea receptors.     

Mechanism of fat-induced attenuation of glucose-induced insulin secretion from mouse pancreatic islets

In order to investigate the mechanism behind fat-induced inhibition of glucose-induced insulin secretion a selection of enzymes that may participate in regulation of pancreatic islet glucose oxidation was studied in islets isolated from mice that had been fed on a laboratory chow diet or on a high-fat diet for 10–12 weeks. At 20 mmol/L glucose production of ¹⁴CO₂ from [U-¹⁴C]-glucose was decreased 50% in islets from fat-fed mice. At 3.3 mmol/L glucose the glucose oxidation rate was similar in the two groups. The fatinduced decrease in glucose oxidation rate was correlated with a 35% decrease in the maximal glucokinase activity. The K_m for glucose was unchanged. No differences between the diet groups were found in the activities of hexokinase, phosphofructo-1-kinase, glucose 6-phosphatase or mitochondrial glycerophosphate dehydrogenase. After preincubation with 20 mmol/L glucose the activity of cytosolic Ca²⁺-independent as well as Ca²⁺-dependent phospholipase A₂ was unchanged by fat-feeding. However, the activity of lysophospholipase was significantly increased by fat feeding, which may result in lowered concentrations of islet lysophosphatidylcholine (lysoPC). It is concluded that in fat-induced diabetic animals a decrease in islet glucokinase may contribute considerably to the decrease in islet glucose oxidation rate. Furthermore, the study raises the possibility that changes in islet lysoPC may contribute to the fat-induced attenuation of glucose-induced insulin secretion. Received: 1 February 1999 / Accepted: 19 May 1999     

Regulation,perturbation, and correction of metabolic events in pancreatic islets

The physiological regulation of nutrient catabolism in islet cells, its perturbation in non-insulin-dependent diabetes mellitus, and the tools available to compensate for such a perturbation are reviewed. In terms of physiology, emphasis is placed on the relevance of glucokinase to hexose-induced insulin release, protein-to-protein interaction and enzyme-to-enzyme channelling, and the preferential stimulation of mitochondrial oxidative events in glucose-stimulated B-cells. In terms of pathology, attention is drawn to the deficiency of FAD-linked mitochondrial glycerophosphate dehydrogenase. Last, as far as therapeutic aspects are concerned, the potential usefulness of hypoglycemic sulfonylureas and meglitinide analogs, adenosine analogs, non-glucidic nutrients, and GLP-1 is underlined.Invited lecture presented during the 6th International Milano Meeting on Diabetes held in Milan on 21–23 March, 1996     

Is type 2 diabetes due to a deficiency of FAD-linked glycerophosphate dehydrogenase in pancreatic islets?

The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (m-GDH) is thought to play a key role in the glucose-sensing mechanism of the insulin-producing B-cell. It catalyses a rate-limiting step of the glycerol phosphate shuttle in pancreatic islets. Its activation by Ca²⁺ accounts for the preferential stimulation of oxidative glycolysis and, hence, pyruvate oxidation in glucose-stimulated islets. Reduced activity of m-GDH was recently observed in islet, but not liver, homogenates from rats injected with streptozotocin during the neonatal period and in two models of inherited diabetes, i.e. GK rats anddb/db mice. In the streptozotocin-injected and GK rats the m-GDH islet defect coincided, in intact islets, with an abnormally low ratio between oxidative and total glycolysis. Decreased activity of m-GDH in T-lymphocytes was also observed in 12 of 32 type 2 (non-insulin-dependent) diabetic patients, but only once among 26 other subjects including 11 healthy volunteers, 9 non-diabetics and 6 patients with either type 1 (insulin-dependent) or symptomatic diabetes. In the T-lymphocytes of type 2 diabetics the m-GDH deficiency occasionally coincided with an abnormally high ratio between glutamate-pyruvate and glutamate-oxaloacetate transaminase activities, as also observed in islets from streptozotocin-injected or GK rats. It is speculated that an islet m-GDH defect could represent a far from uncommon factor contributing to the pathogenesis of type 2 diabetes mellitus.     

Lymphokine release during co-culture of human lympho-mononuclear cells and fresh or cultured human,porcine and bovine pancreatic islets

In this study we evaluated whether isolated human (HI), porcine (PI) and bovine (BI) islets, either fresh (Fr) or cultured for 4 weeks (4w) affect cytokine release from human lymphomononuclear cells (LMC) differently. We prepared LMC from peripheral blood by density gradient purification and co-cultured 1×10⁶ LMC for 24 h with 100 hand-picked islets, either within 48 h of isolation or after culture for 4 weeks. Soluble interleukin-2 receptor (IL-2R), interferon-gamma (IFN), interleukin-4 (IL-4) and interleukin-10 (IL-10) were measured by sandwich enzyme-linked immunoadsorbent assay. Compared with controls (Ctrl, LMC without islets), Fr-HI, Fr-PI and Fr-BI caused a similar increase of IL-2R and IFN release, whereas 4w-HI and 4w-BI did not lead to any significant production of these two cytokines. IL-10 concentrations increased with Fr-PI and Fr-BI, but not with Fr-HI, and no major effect of the 4-week culture was seen. IL-4 levels were below the detection limit of the method used in these experiments. Thus, fresh allo- and xeno-islets caused a similar increase of the release of cytokines known to be markers of Th1 activation, whereas the release of IL-10, a marker of Th2 activation, increased with xeno-, but not with allo-islets; culturing the islets for 4 weeks decreased Th1, but not Th2 activation.     

Structural changes of pancreatic islets in genetically obese rats

Summary Light and electron microscopic observations were performed on pancreatic islets from genetically obese rats, (Zucker, fatty), from 5 to 52 weeks of age. At 5 weeks of age, islets were moderately hypertrophied. After that age, hypertrophy of islets became more prominent, until 24 weeks of age, with accompanying degranulation of B cells. The plasma insulin level also continued to increase during this period, but the glucose level was normal. Degranulated B cells contained a highly developed Golgi complex, numerous vesiculated, granular, endoplasmic reticulum and a small number of secretory granules, but no glycogen deposits. Emiocytosis and microtubule formation were very remarkable with these B cells. Frequently, mixed or intermediate cells, such as exocrine-endocrine or ductural-endocrine cell, were observed in pancreas with hypertrophied islets. At 52 weeks of age, both the plasma insulin and triglyceride levels decreased. In the pancreas, there were observed proliferation of fibrous tissue and well granulated B cells in hypertrophied islets. Hence, in fatty rats, pancreatic islets were in an active state during the period of development of obesity and hyperlipaemia (from 5 to 24 weeks of age). These correlates of obesity and hyperinsulinism disappeared at 52 weeks of age.     

Adenine nucleotide pattern in rat pancreatic islets exposed to nutrient secretagogues

The effects of d-glucose, d-mannose, d-galactose, d-glyceraldehyde, pyruvate, l-lactate, 2-ketoisocaproate, l-leucine, and/or l-glutamine on the ATP and ADP content of rat isolated pancreatic islets were reevaluated in order to compare changes evoked by these nutrient secretagogues in the islet ATP content and ATP/ADP ratio to their effects upon insulin release. Although being compatible with the fuel concept for nutrient-stimulated insulin secretion, the results of this study also argue against the monolithic view that the adenine nucleotide pattern in islet cells represents the sole coupling factor between metabolic and more distal events in the process of nutrient-stimulated insulin release.     

Differential inhibitory action of cationic amino acids on protein synthesis in pancreatic rat islets

Summary The effect of cationic amino acids, i.e. L-arginine and L-lysine, on protein synthesis in isolated rat islets of Langerhans has been investigated. Except for prosomatostatin, the formation of islet proteins is strongly depressed by these amino acids. This effect can be demonstrated within a few minutes and is rapidly reversible. For proglucagon, efficient concentrations of arginine are in the range of 1 to 10 mmol/l. The sensitivity of proinsulin formation to arginine is glucose-dependent: at 2.5 mmol/l, inhibitory concentrations of arginine are 10-fold lower than in the case of proglucagon. High glucose (20 mmol/l) almost completely protects proinsulin synthesis from this inhibition. The proteolytic conversion steps in hormonal precursor processing are not influenced by cationic amino acids as studied in intact islets and in a cell-free translational system. It is concluded that arginine and lysine inhibit protein synthesis in islet cells at the translational level. The release of these amino acids by prohormone conversion may exert a feed-back control on proinsulin formation that is modulated by glucose.     

Effects of L-leucine on the insulin production,oxidative metabolism and mitochondrial ultrastructure of isolated mouse pancreatic islets in tissue culture

Summary This study was performed to evaluate whether L-leucine is able to relieve the structural and functional alterations previously described in pancreatic islets exposed in vitro for a prolonged time to a subnormal glucose concentration (3.3 mM). It was found that both the impairment of secretion and the decreased rate of biosynthesis of insulin characteristic, of islets cultured for one week in 3.3 mM glucose were prevented by adding 15 mM L-leucine to the culture medium. Furthermore, the rates of tritiated water production and glucose or leucine oxidation were significantly enhanced after culture in the presence of L-leucine. The rate of DNA synthesis as estimated by the incorporation of tritiated thymidine was, however, unchanged by the presence of L-leucine in the culture medium. Leucine cultured islet cells displayed ultrastructural signs of high functional activity. A detailed morphometric examination revealed fewer but hypertrophic mitochondria. The present results suggest that L-leucine can replace glucose in several respects as a long-term stimulus of the pancreatic B-cells, possibly by acting as a metabolic substrate.Presented in part at the Congresses of the European Association for the Study of Diabetes, Munich, 1975 and Helsinki, 1976     

Role of infiltrating T cells for impaired glucose metabolism in pancreatic islets isolated from non-obese diabetic mice

Summary Pancreatic islets isolated from non-obese diabetic (NOD) mice, all of which have insulitis, exhibit an impaired glucose metabolism. In order to investigate the role of infiltrating lymphocytes for this altered metabolism, we injected 12- to 13-week-old female NOD mice with monoclonal antibodies directed against either the -T cell receptor, CD4⁺ or CD8⁺ T cells. Control NOD mice were injected with normal rat IgG or with the vehicle (phosphate buffered saline) alone. Injection of the three different monoclonal antibodies markedly reduced the mononuclear cell infiltration. An intravenous glucose tolerance test showed no differences between the groups. Islet insulin release in response to glucose was similar in all groups. In contrast, islets isolated from the control NOD mice with insulitis showed a high basal (1.7 mmol/l glucose) glucose oxidation rate and a small increase in the glucose oxidation rate in response to a high glucose concentration (16.7 mmol/l glucose). The monoclonal antibodies counteracted the elevated basal glucose oxidation rate of the islets. Parallel studies of stimulated mononuclear cells suggested that the contribution of glucose oxidized by islet-infiltrating lymphocytes could only partially explain the observed alterations in NOD mouse islet metabolism. Culture of islets obtained from NOD mice in the presence of the cytokine interleukin-1 induced a similar pattern of glucose metabolism as seen earlier in IgG or phosphate-buffered saline treated control NOD mice. In conclusion, alterations in the glucose oxidation rates seem to be an early sign of disturbance in islets isolated from NOD mice. These early alterations in glucose metabolism can be reversed in vivo by monoclonal antibodies directed against effector lymphocytes. This suggests that the infiltrating mononuclear cells can induce reversible alterations in pancreatic Beta-cell function which may precede impaired insulin secretion, Beta-cell destruction and overt diabetes mellitus.     

Size of pancreatic islets of Langerhans: a key parameter for viability after cryopreservation

Abstract. Large amounts and excellent viabilities of pancreatic islets are prerequisites for recent advances in islet transplantation. Cryopreservation has been shown to enlarge transplanted cell mass, but has been accompanied by reduced viability. In this study rat pancreatic islets were differentiated into small (<200 µm), medium (200–400 µm) and large (>400 µm) categories and their susceptibilities to different freezing conditions were evaluated: concentration of cryoprotectant (0.7–3.1 M), equilibration (15 vs. 45 min, 22° C vs. on ice) and post-thaw removal of cryoprotectant (15 vs. 30 min, stepwise vs. one-step). The most prominent finding was a negative correlation between islet size and viability observed in non-frozen islets to a minor degree (r=-0.44) and significantly enhanced after cryopreservation (r<-0.8). The concentration of cryoprotectant showed the most significant influence on viability affecting small, medium and large islets. Different techniques of equilibration with the cryoprotectant resulted in significant changes of islet viability of medium islets, whereas small and large islets were unaffected. For different techniques of removal of the cryoprotectant, no significant influence on viabilities was found. We conclude that large islets represented a highly susceptible population concerning damage due to cryopreservation.     

Culture of mouse pancreatic islets in different glucose concentrations modifies B cell sensitivity to streptozotocin

Summary There have previously been divergent data published regarding the effects of glucose on the diabetogenic effects of streptozotocin. In order to further explore this issue, two separate sets of experiments were performed. In the first, mouse pancreatic islets were maintained in culture for 3 days at different glucose concentrations (5.6,11.1 and 28 mmol/l) and then exposed to streptozotocin. After another 3 days in culture at 11.1 mmol/l glucose, the B cell function was evaluated by measurement of glucose-stimulated insulin release, the number of islets recovered after culture, and the islet DNA and insulin contents. In the second group of experiments islets were first maintained in culture at 11.1 mmol/l glucose, then treated with streptozotocin and subsequently cultured for 6 days at the different glucose concentrations given above. It was found that islets maintained in a medium containing 28 mmol/l glucose before or after streptozotocin exposure showed less signs of damage than islets cultured in 11.1 mmol/l glucose. A similar, but less pronounced, de creased sensitivity to streptozotocin was found in islets precultured in 5.6 mmol/l glucose, in comparison with those islets cultured in 11.1 mmol/l glucose. Culture at 5.6 mmol/l glucose just after streptozotocin treatment did not induce any improvement in islet survival or function. It is suggested that the increased damage induced by streptozotocin to islets precultured at 11.1 mmol/l glucose, in comparison with 5.6 mmol/l glucose, can be related to the fact that an increased metabolic activity of B cells render them more susceptible to the toxin. The improved preservation of islets cultured at 28 mmol/l glucose before or after streptozotocin treatment may reflect an additional effect of glucose, i. e. activation of defense mechanisms in the B cells against cytotoxins.     

Glycogen accumulation in rat pancreatic islets

Under conditions of sustained hyperglycemia, glycogen accumulates in pancreatic islets, but not so in acinar pancreatic cells. Advantage conceivably could be taken from such a situation in the perspective of the noninvasive imaging of the endocrine pancreas. The present experiments aim, therefore, at characterizing the time course for glycogen accumulation in pancreatic islets cultured at a high concentration (30 mM) of d-glucose in the presence of tracer amounts of either d-[U-¹⁴C]glucose or 2-deoxy-2 [¹⁸F]fluoro-d-glucose. The ¹⁴C-labeled glycogen content of the cultured islets increased with time (150 min to 72 h), exceeded that found in acinar tumoral cells, and did not decrease over 60 min of incubation at 30 mM d-glucose in the absence of d-[U-¹⁴C]glucose. Glycogenolysis was observed, however, when the concentration of d-glucose was decreased to 2.8 mM and, in such a case, was further enhanced by forskolin and theophylline. Such a glycogenolysis concided with the generation of ¹⁴CO₂ from radioactive intracellular precursors and alteration of the B-cell secretory response to d-glucose. The radioactive glycogen content was higher in islets exposed to 2-deoxy-2-[¹⁸F]fluoro-d-glucose than d-[U-¹⁴C]glucose. Prior exposure of the islets to streptozotocin suppressed the accumulation of glycogen during their subsequent culture at high d-glucose concention. These findings may help to define the experimental conditions optimal for the labeling and accumulation of islet glycogen in vivo.     

胰岛微循环与胰岛功能

胰岛微循环是维持胰岛结构和功能的基础,每个胰岛由1～5个动脉供应血液.动脉分支为毛细血管后在胰岛内形成血管球框架,胰岛细胞位于血管球内,从血管中摄取营养物质及氧气.胰岛微循环受局部合成的生物活性分子、神经系统及胰岛分泌激素等调节,与胰岛功能关系密切,参与糖尿病胰岛功能障碍发生发展过程,是糖尿病防治研究的新视角.