Fluctuations of alpha cell calcium, potassium and sodium during amino acid perifusion of rat pancreatic islets

Perifusion of rat pancreatic islets with a physiologic, 6-mM amino acid mixture resulted in typical acute and second phase glucagon secretion over 30 min. At various intervals, islets were acutely fixed and processed for scanning electron microscopy, identification of alpha cells, and measurements of single alpha cell content of calcium (Ca), potassium (K) and sodium (Na) with energy-dispersive x-ray analysis. Biphasic glucagon secretion was attended by corresponding biphasic Ca accumulation and a reciprocal, biphasic suppression of K content and acute phase suppression of Na in alpha cells. All secretory and cellular events were preceded by an evanescent upward spike in alpha cell K at 1 min. These results indicate that alpha cell glucagon secretion in response to amino acid mixtures may be initiated by a K signal and is coupled subsequently to phasic changes in alpha cell Ca content. Fluctuations of alpha cell K and Na appear to relate inversely to Ca, suggesting that transmembrane fluxes of the three cations are interrelated.     

Tritiated taurine handling by isolated rat pancreatic islets

A gating of volume-sensitive anion channels may participate in the depolarization of the plasma membrane caused by high concentration of d-glucose in insulin-producing B-cells of the endocrine pancreas. The efflux of tritiated taurine from prelabeled cells is currently used to assess changes in the activity of such channels. The handling of [1,2-³H]taurine by isolated rat pancreatic islets was therefore investigated. The net uptake of [1,2-³H]taurine was found to represent a concentration-, time-, and temperature-dependent process. It was progressively increased in the range of d-glucose concentrations between 2.8 and 8.3 mM, but no further increase was observed at 16.7 mM d-glucose. Over 15 min incubation, the efflux of radioactivity from prelabeled islets was inhibited by MK571 (1.0 mM). It was increased in response to hypoosmolarity both in the presence and absence of extracellular Na⁺. Whether in salt-balanced or Na⁺-deprived media, the efflux of radioactivity from prelabeled islets increased in response to a rise in d-glucose concentration from 2.8 to 5.6 or 8.3 mM, but decreased when the concentration of the hexose was further increased from 8.3 to 16.7 mM. In perifused islets, however, the radioactive efflux from prelabeled islets was inhibited, in a concentration-related manner, when islets first deprived of d-glucose for 45 min were then exposed to 2.8, 5.6, or 16.7 mM d-glucose. Likewise, in prelabeled and perifused islets first exposed for 45 min to 4.0 mM d-glucose, a later rise in hexose concentration to 8.3 mM failed to affect significantly effluent radioactivity, while an increase in hexose concentration from 4.0 to 16.7 mM inhibited the radioactive outflow. In these perifusion experiments, the rise in d-glucose concentration provoked the expected changes in insulin output. The findings obtained in islets examined immediately after preincubation in the presence of [1,2-³H]taurine are consistent with the presence of volume-sensitive anion channels in islet cells and with a gating of such channels in response to a rise in d-glucose concentration from 2.8 to 5.6–8.3 mM. However, the radioactive fractional outflow rate from prelabeled islets seems to reach its highest value at about 8.3 mM d-glucose, being unexpectedly decreased at a higher concentration (16.7 mM) of the hexose. In conclusion, the pleiotropic effects of d-glucose upon tritiated taurine outflow from prelabeled rat islets, which could conceivably be ascribed to differences in the handling of this amino sulfonic acid by distinct islet cell types, indicates that the present approach is far from optimal to characterize unambiguously the regulation by the hexose of volume-sensitive anion channel activity in insulin-producing islet cells.     

Beta-endorphin inhibits insulin secretion from isolated pancreatic islets

Intravenous administration of small doses of beta-endorphin causes immediate suppression of basal and glucose-stimulated insulin secretion in normal rabbits. The purpose of the present study was to determine if beta-endorphin directly inhibits glucose-stimulated insulin secretion from rabbit pancreatic islets. Islets were isolated from male New Zealand White rabbits and perifused for 1 h with medium containing 100 mg/dl glucose (M100) followed by a 1-h challenge with medium containing 300 mg/dl glucose (M300) with or without beta-endorphin and/or the specific opioid antagonist naloxone. Samples were collected every 5 min during the last 30 min of the baseline perifusion with M100 and during the 1-h challenge with the stimulatory concentration of glucose (M300). Total insulin secretion for each 1-h period was calculated by adding the areas under the curves for twice the 30-min baseline period and for the 1-h challenge period. The mean +/- SE area for the control islets during perifusion with M100 was 5.9 +/- 0.8 microU/islet.h. M300 stimulated a 4.2-fold increase in the amount of insulin secreted (24.5 +/- 3.6 microU/islet.h). The stimulated rate of insulin release was sustained throughout the 1-h test period with M300, averaging 0.42 +/- 0.02 microU insulin/islet.min. beta-Endorphin inhibited glucose-stimulated insulin secretion in a concentration-dependent manner. Maximal suppression of insulin secretion to a level well below the baseline secretion rate was produced by 300 nM beta-endorphin (1.9 +/- 0.3 microU/islet.h). The first 15 min of glucose-stimulated insulin secretion was 6 times less sensitive to the inhibitory effect of beta-endorphin than was the next 45 min. The concentrations of beta-endorphin causing 50% inhibition of glucose-stimulated insulin secretion (IC50) for the 5- to 15-, 20- to 60-, and 5- to 60-min intervals were 1.96, 0.35, and 0.57 nM, respectively. Naloxone (3 microM) had no effect on glucose-stimulated insulin secretion, but partially antagonized the inhibitory effect of 30 nM beta-endorphin (10.2 +/- 2.9 microU/islet.h naloxone plus beta-endorphin vs. 2.6 +/- 1.1 microU/islet.h beta-endorphin; P less than 0.05). These data demonstrate that beta-endorphin, at low concentrations, has a direct inhibitory effect on insulin secretion, and they support the idea that a naloxone-sensitive beta-endorphin-binding component is present in pancreatic islets.     

Successful intra-splenic transplantation of syngeneic and allogeneic isolated pancreatic islets

Summary Adult rat islets harvested by the collagenase digestion/Ficoll separation technique were injected into the splenic pulp in 9 syngeneic (Lewis-Lewis and WaGWaG) and 13 allogeneic [(DA X Lewis) F1Lewis] experiments. Normal serum glucose levels and 24 hour urine volumes were restored in all 9 syngeneic recipients and in 11 out of the 13 allogeneic recipients in a mean of 3.3 days. Splenectomy performed on 3 of the 9 syngeneic recipients 110–178 days after transplantation resulted in a prompt return to the diabetic state. In all the remaining syngeneic recipients, normal values have persisted for the current period of observation of 6 months. In 5 untreated allogeneic recipients, rejection occurred in a mean of 5.2 days. The administration of a short course of ALS (1 ml I. P. days —1, 1, 3 and 5) to the remaining 6 animals greatly prolonged graft survival with all animals remaining normoglycaemic for at least 4 weeks. These results were not significantly different from those recorded in comparable groups of intra-portal allogeneic islet recipients.This work was supported by an MRC programme grant and the Wellcome Foundation     

血管紧张素Ⅱ与人离体胰岛功能研究

目的探讨分离纯化的人胰岛表面血管紧张素Ⅱ1型受体(AT1)的存在与否,了解血管紧张素Ⅱ对人胰岛分泌胰岛素功能的影响。方法对分离纯化后的胰岛进行AT1受体和胰岛素的免疫荧光双标法检测;使用不同剂量血管紧张素Ⅱ探讨其对人胰岛胰岛素释放反应的影响。结果免疫荧光双标记染色胰岛AT1受体和胰岛素均呈强阳性;血管紧张素Ⅱ对高糖刺激胰岛素分泌具有剂量依赖的抑制效应,用AT1受体拮抗剂预处理胰岛能拮抗这种抑制作用。结论分离纯化的人胰岛存在AT1受体,血管紧张素Ⅱ能够通过AT1受体直接抑制胰岛的胰岛素释放反应。     

Chronic exposure to glibenclamide impairs insulin secretion in isolated rat pancreatic islets

We investigated the effect of 24 h exposure to 100 nmol/l glibenclamide on insulin secretion in isolated rat pancreatic islets. The insulin content was similar in control islets and in islets preincubated with 100 nmol/l glibenclamide for 24 h. In islets preexposed to glibenclamide: 1) the subsequent response to a maximal glibenclamide stimulatory concentration (10 mumol/l, 1 h at 37 C) was greatly reduced in comparison to control islets (0.69 +/- 0.20% vs 2.16 +/- 0.41%; mean +/- SE; n = 14; p less than 0.001); 2) the response to 100 mumol/l tolbutamide stimulation was also reduced (0.55 +/- 0.15% vs 2.38 +/- 0.44%; n = 8; p less than 0.001); 3) the response to 16.7 mmo/l glucose, both in the presence or in the absence of 1 mmol/l IBMX, a phosphodiesterase inhibitor, was also diminished by about 50% (1.79 +/- 0.39% vs. 3.22 +/- 0.42%; n = 14, p less than 0.001). In glibenclamide pretreated islets, blunted responses to stimuli were confirmed also by dynamic studies using a perifusion system. The effect of glibenclamide preincubation was fully reversible: when islets cultured in the presence of glibenclamide were transferred to a glibenclamide-free medium for further 24 h, insulin release in response to glibenclamide stimulation returned to control values. We conclude that prolonged exposure of rat pancreatic islets to glibenclamide induces a reversible desensitization to a variety of metabolic stimuli. The inhibition by prolonged glibenclamide exposure of a common pathway in the mechanism of insulin release is one possible explanation for these results.     

Fluorescence of oxidized flavoproteins from perifused isolated pancreatic islets

In perifused pancreatic islets, the fluorescence of oxidized flavoproteins (FAD) was recorded continuously. Elevation of glucose concentration in the medium form 0 or 5 mM to 20 mM led to decrease in FAD-fluorescence beginning 10 sec after change of medium. L-leucine (10 mM), (+/-)-B-BCH (20 mM) and alpha-ketoisocaproic acid (10 mM) caused typical kinetics of FAD-fluorescence decrease. The results are interpreted to indicate rapid changes of the functional state of B-cell mitochondria induced by the above-mentioned stimulators of insulin release.     

GLP-1可拮抗Ghrelin对离体大鼠胰岛分泌胰岛素的抑制

胰岛素是机体调何血糖水平的主要激素。Ghrelin主要由胃分泌,可增进食欲,促进能量正平衡,调节血糖水平,诸多研究证明,Ghrelin可明显抑制葡萄糖刺激的胰岛豢分泌。     

Hyperinsulinism induced with glucocorticoid--a study on using a perifusion system of isolated islets in rats (author's transl)

In order to elucidate the mechanism of hyperinsulinism following a treatment with glucocorticoid, insulin secretion induced with glucose or tolbutamide was investigated by a perifusion experiment on isolated islets of rats. The results are summarized as follows: 1. The fasting blood glucose level was significantly higher on the 2nd day (174.0 +/- 11.8 mg/dl) and 3rd day (179.6 +/- 9.5 mg/dl) in the glucocorticoid treated rats, than it was in the control rats (129.0 +/- 12.0 mg/dl). The serum insulin levels began to increase from the first day following the glucocorticoid treatment (17.2 +/- 1.3 microU/ml in the control rats, 27.6 +/- 2.1 microU/ml on the 1st day, 32.4 +/- 3.9 microU/ml on the 2nd day, and 34.5 +/- 1.4 microU/ml on the 3rd day). 2. The insulin content of the islets decreased with the glucocorticoid treatment (765.6 +/- 34.5 microU/islet in the control rat, 576.6 +/- 25.0 microU/islet on the 1st day, 629.2 +/- 36.9 microU/islet on the 2nd day, and 482.0 +/- 43.5 microU/islet on the 3rd day). 3. In the perifusion experiment, a biphasic pattern of insulin secretion was demonstrated with the stimulation of glucose in the control and glucocorticoid treated rats. A remarkable enhancement of insulin secretion was observed by the stimulation of 100 mg/dl glucose. The amount of insulin secretion at the first phase (up to 7 min. after the glucose stimulation) was 2.9 +/- 0.5 microU/islet on the 1st day, 2.7 +/- 0.3 microU/islet on the 2nd day and 3.8 +/- 0.1 microU/islet on the 3rd day; these amounts were high compared with that of 1.8 +/- 0.1 microU/islet in the control rat. The amount of insulin secretion at the second phase (8 to 60 min. after the glucose stimulation) was 28.5 +/- 2.5 microU/islet on the 1st day, 37.1 +/- 3.3 microU/islet on the 2nd day and 41.3 +/- 1.8 microU/islet on the 3rd day; these amounts were higher when compared with that of 24.7 +/- 0.7 microU/islet in the control rat. 4. The monophasic insulin secretion from isolated islets by the stimulation of tolbutamide was enhanced with the treatment of glucocorticoid. These results indicate that glucocorticoid directly enhances insulin secretion from the pancreatic islets at the physiological concentration of blood glucose, which seems to be an important factor in the occurrence of hyperinsulinemia associated with glucocorticoid therapy.     

Glucose decreases extracellular adenosine levels in isolated mouse and rat pancreatic islets

The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca²⁺ suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.     

Glucose decreases extracellular adenosine levels in isolated mouse and rat pancreatic islets

The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca²⁺ suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.     

A method for enzymatic dissociation of cells from isolated pancreatic islets

Summary An enzymatic method for isolation of single cells from the islets of Langerhans is described. The isolated cells appeared well preserved and survived for at least 7 days when maintained in culture. The dry mass of the isolated islet cells was found to be decreased 30 min after administration of alloxan to obese-hyperglycemic mice. Isolated individual islet cells from obese-hyperglycemic mice had a higher dry mass than those from their lean litter mates. Traduzione a cura di G. U.     

Influence of cholecystokinin on insulin output from isolated perifused pancreatic islets

The C-terminal eight-amino acid derivative of CCK, sulfated on the tyrosine residue (CCK8S), stimulated a dose-dependent biphasic pattern of insulin secretion from isolated perifused islets in the presence of 7 mM glucose. It was without any effect if glucose were absent from the medium or maintained at 4 mM. The response to CCK8S was readily reversible and dependent on the presence of extracellular calcium. While CCK8S did not increase glucose usage rates above those noted with 7 mM glucose alone, inclusion of the metabolic inhibitor 2-deoxyglucose lowered glucose usage rates to values obtained with 3-5 mM glucose and abolished the influence of CCK8S on insulin output. Removal of the metabolic inhibitor restored the secretory response. N-Acetylglucosamine (15 mM) or glyceraldehyde (2.5 mM) substituted for glucose and permitted CCK8S to evoke secretion. The nonsulfated eight-amino acid derivative of CCK, CCK8, provoked insulin secretion in the presence of 7 mM glucose, but only at 10-100 times greater levels than CCK8S. CCK4 (1 microM) did not influence insulin output in the presence of 7 mM glucose. On an equimolar basis, CCK8S was significantly more effective than gastric inhibiting polypeptide in augmenting insulin output. The results support a role for CCK8S in the regulation of insulin levels in vivo.     

Inhibition by kynurenine metabolites of proinsulin synthesis in isolated pancreatic islets

Summary The effect of kynurenine metabolites on insulin biosynthesis was investigated in isolated pancreatic islets of the rat. Both quinaldic acid and 8-hydroxyquinaldic acid were found to produce significant inhibition of the proinsulin synthesis. However, the conversion process of proinsulin to insulin in the islet was not affected by these kynurenine metabolites. Furthermore, the inhibitory effect of these end-metabolites of kynurenine was characterized by preferential inhibition of proinsulin synthesis as distinct from non-insulin protein synthesis in the islet. In contrast to the significant inhibitory effect of quinaldic acid and 8-hydroxyquinaldic acid on proinsulin synthesis, xanthurenic acid and kynurenic acid were far less effective, and L-tryptophan, L-kynurenine, 3-hydroxyanthranilic acid and quinolinic acid showed little ability to inhibit proinsulin synthesis in islets. Preliminary data from this work were presented at the 9th Congress of the IDF, New Delhi, India (Excerpta Medica Foundation Congress Series no. 400; p. 8) and at the 2nd International Meeting on Tryptophan Metabolism, Madison/Wisc., U.S.A., August 10–12, 1977. On leave from the Department of Internal Medicine, Kanazawa University School of Medicine, Kanazawa, Japan.     

Ghrelin对离体大鼠胰岛分泌胰岛素的影响

目的 观察Ghrelin对离体大鼠胰岛分泌胰岛素的影响.方法 将分离纯化的大鼠胰岛置于含不同浓度葡萄糖和Ghrelin的孵育液中孵育1h,应用放射免疫分析法测定孵育液中胰岛素浓度.结果 葡萄糖浓度＞5.6 mmol/L时,浓度≥10 nmol/L的Ghrelin对胰岛素分泌有明显抑制作用(P<0.05).结论 高浓度Ghrelin可显著抑制高浓度葡萄糖诱导的胰岛素分泌.     

Feedback inhibition of insulin on insulin secretion in isolated pancreatic islets

We have examined the effect of exogenous insulin on secretion vesicle margination and secretion vesicle lysis in isolated perifused rat pancreatic islets. Recruitment of somatostatin (SRIF) receptors to the plasma membrane was used as a marker of secretion vesicle margination, whereas insulin release reflected the process of secretion vesicle lysis. A newly designed perifusion protocol allows one to interrupt intermittently either secretion vesicle margination or secretion vesicle lysis. Islets were initially perifused with glucose (30, 100, 165, 200, or 300 mg/dl) in the presence of sodium isethionate. Sodium isethionate inhibits secretion vesicle lysis, but not the recruitment of SRIF receptors. Thus, the margination of secretion vesicles to the surface membrane continued without their lysis. Sodium isethionate was then removed, and islets were challenged with 400 microM isobutylmethylxanthine (IBMX). In the islets perifused with high glucose concentrations, IBMX lysed a greater number of vesicles and caused enhanced release of insulin. The presence of exogenous insulin during the initial phase of secretion vesicle margination did not affect subsequent IBMX-induced insulin secretion from the islets perifused with low glucose concentrations (30 or even 100 mg/dl). When the glucose concentration was increased to 165, 200, or 300 mg/dl, insulin significantly diminished IBMX-induced insulin release. In separate experiments, increasing concentrations of insulin (50, 100, and 200 microU/ml) reduced glucose-induced recruitment of SRIF receptors in a dose-dependent manner. Our observations strongly suggest the existence of a well balanced relationship between ambient glucose and insulin concentrations in terms of their positive and negative feedback actions on insulin release. Their influences seem to be exerted at the level of secretion vesicle margination at the plasma membrane.     

4-deoxypyridoxine improves the viability of isolated pancreatic islets ex vivo

The successful islet transplantation, for the treatment of type 1 diabetes, depends on the quantity and the quality of transplanted islets. Previously, it has reported that the significant loss of isolated islet mass could be prevented by sphingolipid metabolite, sphinogosine 1-phophate (S1P).

This study was performed to elucidate whether the beneficial effects of S1P maintaining isolated pancreatic islets ex vivo are mimicked by modulation of intracellular S1P. We tested the in vitro effect of various agents that modulate intracellular S1P levels in insulinoma cell lines and isolated islets to compare their anti-apoptotic effects with that of S1P.

As results, we discovered that 4-deoxypyridoxine (DOP), which inhibits the degradation of intracellular S1P by inhibiting S1P lyase (SPL) activity, minimized the chemically induced apoptosis of insulinoma cell lines as S1P did. Also, supplementation of DOP in the culture media protected the regression of isolated islets that have been maintained ex vivo at least for 18 h providing the evidence of increasing viability of isolated islets with DOP, which impaired SPL activity.

In conclusion, these results suggest that the application of SPL inhibitors could be considered as a supplement for the maintenance of viable islets isolated from donor sources in the process of islet transplantation.