Investigations on isolated islets of Langerhans in vitro. Influence of temperature changes during preparation on some parameters of insulin metabolism.

Pancreatic islets of Wistar rats were prepared by digestion with collagenase and then washed and isolated at three different temperatures (4, 22 and 37 degrees C). The efficiency of washing with regard to proteolytic and collagenolytic activities in the wash buffer was not affected by the temperatures used. The islet thiol:protein-disulphide oxidoreductase activity (EC 1.8.4.2) was apparently unchanged, whereas washing temperatures lower than 37 degrees C resulted in a diminished insulin content. The insulin secretion of islets, isolated at 4 degrees C, is reduced in response to glucose without changing the sigmoidal shape of dose-response curve.     

Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships

The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.     

Stimulation of insulin secretion from mouse pancreatic islets, maintained in tissue culture, by the pituitary neurointermediate lobe of the genetically obese mouse (ob/ob)

The rapid stimulation of insulin release by a perifusate from the pituitary neurointermediate lobe of the $\text{ob}\text{ob}$ mouse has been demonstrated by perifusing collagenase prepared mouse islets maintained for 48 hours in tissue culture. The maximal stimulation occurred in about 2 minutes and insulin secretion remained slightly above basal levels for 10 minutes. Freshly prepared collagenase islets showed no response to the pituitary factor and after 24 or 72 hours in culture there was a significant but reduced response compared to the 48 hour cultured islets.     

Effect of the pancreatic digestion with liberase versus collagenase on the yield, function and viability of neonatal rat pancreatic islets

Many obstacles hinder the clinical application of pancreatic islet transplantation as a cure for diabetes mellitus. One of them is the suitable isolation method of sufficient number of healthy islets for transplantation. In this context, liberase enzyme was developed as a purified form of the traditional collagenase. It was the aim of this study to investigate the effect of liberase-digestion on the yield, function and viability of neonatal rat islets, and to compare the new enzyme with the collagenase. Glucose-stimulated insulin secretion was measured as indication of the function, insulin content as indication for the synthetic activity of islet cells and DNA as an indication of cell viability. The results showed no difference between islets isolated either with collagenase or liberase. Glucose stimulated similarly the insulin secretion in both. Stimulation index tended, without significance, to be higher (55%) in liberase-isolated islets compared with the collagenase islets (49%). The viability of both was similar. The insulin synthesis (content) tended also to be better in liberase-isolated islets. It could be concluded that liberase could be non-significantly preferred in the isolation of neonatal rat islets in comparison with collagenase.     

Beta cell contact and insulin release

Insulin secretion by intact islets, dispersed islet cells and dispersed cells allowed to reaggregate was compared in perifusion. Although single cells and aggregates showed basal insulin secretion and a prompt response to glucose challenge, basal secretion, peak insulin secretion and total insulin secretion during a 60 minute stimulation were profoundly less than those activities of intact islets. These results suggest that dispersed beta cells are responsive to glucose as a secretagogue, but the magnitude of the response is greatly diminished and not restored by simple cell contact.     

Potassium ions and the secretion of insulin by islets of Langerhans incubated in vitro

1. A method was devised for the isolation of islets of Langerhans from rabbit pancreas by collagenase digestion in order to study the influx and efflux of K(+) in islets during insulin secretion. 2. Glucose-induced insulin release was accompanied by an increased rate of uptake of (42)K(+) by the islets of Langerhans, though this was not the case for secretion in response to tolbutamide. Ouabain significantly inhibited the uptake of (42)K(+) by islet tissue. 3. No significant increase in the rate of efflux of (42)K(+) was demonstrated during active insulin secretion. 4. Slices of rabbit pancreas were incubated in media of different K(+) content, and rates of insulin release were determined. Alteration of the K(+) concentration of the medium between 3 and 8mm had no effect on the rate of insulin release by pancreas slices. However, decrease of the K(+) concentration to 1mm resulted in inhibition of secretion in response to both glucose and to tolbutamide. Conversely, an increase in K(+) concentration increased rates of insulin release in response to both these stimuli. 5. It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes of K(+) in the islets do not appear to be important in the initiation of insulin secretion.     

Decreased nutrient-stimulated insulin secretion in chronically hypoglycemic late-gestation fetal sheep is due to an intrinsic islet defect

We measured in vivo and in vitro nutrient-stimulated insulin secretion in late gestation fetal sheep to determine whether an intrinsic islet defect is responsible for decreased glucose-stimulated insulin secretion (GSIS) in response to chronic hypoglycemia. Control fetuses responded to both leucine and lysine infusions with increased arterial plasma insulin concentrations (average increase: 0.13 +/- 0.05 ng/ml leucine; 0.99 +/- 0.26 ng/ml lysine). In vivo lysine-stimulated insulin secretion was decreased by chronic (0.37 +/- 0.18 ng/ml) and acute (0.27 +/- 0.19 ng/ml) hypoglycemia. Leucine did not stimulate insulin secretion following acute hypoglycemia but was preserved with chronic hypoglycemia (0.12 +/- 0.09 ng/ml). Isolated pancreatic islets from chronically hypoglycemic fetuses had normal insulin and DNA content but decreased fractional insulin release when stimulated with glucose, leucine, arginine, or lysine. Isolated islets from control fetuses responded to all nutrients. Therefore, chronic late gestation hypoglycemia causes defective in vitro nutrient-regulated insulin secretion that is at least partly responsible for diminished in vivo GSIS. Chronic hypoglycemia is a feature of human intrauterine growth restriction (IUGR) and might lead to an islet defect that is responsible for the decreased insulin secretion patterns seen in human IUGR fetuses and low-birth-weight human infants.     

Influence of pilocarpine pretreatment on (pro)insulin biosynthesis of isolated islets of rats.

3[H]-Leucine incorporation into (pro)insulin and insulin secretion was investigated using islets prepared by collagenase digestion from pancreata of rats pretreated with the cholimimetic agent pilocarpine or with saline (controls). Under the influence of pilocarpine pretreatment the [3H]-leucine incorporation into islet proteins with insulin immunoreactivity is enhanced at 6 mM glucose in the incubation medium of the islets but the incorporated radioactivity at 18 mM glucose is independent of the pretreatment of the animals. Only small or no changes were found regarding insulin secretion. It is concluded that an influence of pilocarpine pretreatment should be taken into consideration using such islets for studies on the regulation of (pro)insulin biosynthesis.     

Viability criteria of islets of Langerhans isolated and purified using Ficoll

Rejection of islet allografts is generally explained by immunologic problems, due to both cellular and antibody mechanisms. But another great problem is in the isolation of intact and viable islets of Langerhans: it is necessary to use a good method of pancreas distention, to determine the optimal concentration of collagenase for digestion, to select an effective technique for purifying the islets. This study correlates the morphology of isolated pancreatic islets of rats and dogs with secretion of insulin. The islets are incubated in a perifusion system and are tested during four periods; the glucose concentrations of the perifusion fluid are: 5.5 mM during the initial 70 min. period, 16.5 mM during the second 60 min. period, 5.5 mM during the third 60 min. period and 16.5 during the fourth 50 min. period. This "double glucose stimulation" is a good test of islet viability. The intact, viable isolated islets showed a significant increase of insulin secretion during the two 16.5 mM glucose periods. Damaged islets with some little morphologic alterations after showed a good insulin release during the first glucose stimulation, but a very poor insulin response to glucose during the second stimulation period.     

The CB1 Antagonist Rimonabant Decreases Insulin Hypersecretion in Rat Pancreatic Islets

Type 2 diabetes and obesity are characterized by elevated nocturnal circulating free fatty acids, elevated basal insulin secretion, and blunted glucose‐stimulated insulin secretion (GSIS). The CB1 receptor antagonist, Rimonabant, has been shown to improve glucose tolerance and insulin sensitivity in vivo but its direct effect on islets has been unclear. Islets from lean littermates and obese Zucker (ZF) and Zucker Diabetic Fatty (ZDF) rats were incubated for 24 h in vitro and exposed to 11 mmol/l glucose and 0.3 mmol/l palmitate (GL) with or without Rimonabant. Insulin secretion was determined at basal (3 mmol/l) or stimulatory (15 mmol/l) glucose concentrations. As expected, basal secretion was significantly elevated in islets from obese or GL‐treated lean rats whereas the fold increase in GSIS was diminished. Rimonabant decreased basal hypersecretion in islets from obese rats and GL‐treated lean rats without decreasing the fold increase in GSIS. However, it decreased GSIS in islets from lean rats without affecting basal secretion. These findings indicate that Rimonabant has direct effects on islets to reduce insulin secretion when secretion is elevated above normal levels by diet or in obesity. In contrast, it appears to decrease stimulated secretion in islets from lean animals but not in obese or GL‐exposed islets.     

A Practical Guide to Rodent Islet Isolation and Assessment

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.     

Cyclic AMP-dependent stimulation of somatostatin secretion by isolated rat islets of Langerhans.

Immunoreactive somatostatin is released from islets of Langerhans, isolated from rat pancreas by collagenase digestion, when incubated in an $\text{in vitro}$ system. The rate of somatostatin secretion is independent of extracellular glucose concentration, but is stimulated by addition of 8-Br-cyclic AMP or theophylline.     

L-丙氨酸对小鼠胰岛分泌胰岛素的急性作用及其葡萄糖依赖性研究

探讨L-丙氨酸刺激小鼠胰岛分泌胰岛素的剂量和葡萄糖依赖性。雌性6~10周NMRI小鼠,苯巴比妥腹腔麻醉,应用胶原酶技术消化胰腺分离胰岛,置于RPMI1640培养皿中在37℃培养箱(5%CO2,95%空气)过夜培养。次日在Krebs-Ringer缓冲液中37℃水浴培养箱预培养30 min,分别把单个胰岛小心放入100 L含有不同浓度葡萄糖和不同浓度L-丙氨酸的改良Krebs-Ringer缓冲液37℃水浴培养箱培养60 min,留取50 L上清液进行胰岛素测定。结果:L-丙氨酸在0.1~20mmol.L-1范围促进了葡萄糖刺激的小鼠胰岛的胰岛素分泌,随剂量增大而增强,在低浓度葡萄糖存在的条件下,10 mmol.L-1L-丙氨酸不能刺激小鼠胰岛的胰岛素分泌,在6.7 mmol.L-1及以上葡萄糖存在的条件下,L-丙氨酸能增加葡萄糖诱导的小鼠胰岛分泌胰岛素。本研究显示L-丙氨酸能增加葡萄糖诱导的小鼠胰岛分泌胰岛素,此作用依赖于一定水平葡萄糖的存在。     

Effects of the calcium-channel agonist CGP 28392 on insulin secretion from isolated rat islets of Langerhans.

The rate of insulin secretion from isolated rat islets of Langerhans was affected by a number of dihydropyridine derivatives known to interact with voltage-sensitive Ca2+ channels in excitable cells. The channel antagonists nifedipine and nitrendipine were potent inhibitors of glucose-induced insulin secretion in response to both 8 mM- and 20 mM-glucose, although they did not lower the basal secretion rate observed in the presence of 4 mM-glucose. The Ca2+-channel agonist, CGP 28392, also failed to alter the basal rate of insulin secretion. In the presence of 8 mM-glucose, however, 1 microM-CGP 28392 enhanced the insulin-secretion rate to a value approximately double that with 8 mM-glucose alone. This effect was dose-dependent, with half the maximal response elicited by 0.1 microM-CGP 28392, and full enhancement at 10 microM. The response was rapid in onset, with an increase in insulin secretion evident within 2 min of CGP 28392 infusion in perifused islets. Stimulation of insulin secretion by CGP 28392 was correlated with a rapid enhancement of glucose-stimulated 45Ca2+ uptake into islets cells, and with a transiently increased rate of 45Ca2+ efflux from pre-loaded islets. Stimulation of insulin secretion by CGP 28392 was abolished in the presence of noradrenaline, although under these conditions the rapid stimulation of 45Ca2+ influx induced by CGP 28392 was only partially inhibited. In contrast with these results, when islets were incubated in the presence of 20 mM-glucose, CGP 28392 caused a dose-dependent inhibition of insulin secretion. Half-maximal inhibition required approx. 0.2 microM-CGP 28392, with maximal effects observed at 10 microM. Under these conditions, however, the extent of insulin secretion was still only decreased by about 50%, to a value which was similar to that seen in the presence of 8 mM-glucose and CGP 28392. These results suggest that dihydropyridine derivatives can alter the activity of voltage-dependent Ca2+ channels in islet cells, and are consistent with the possibility that gating of these channels plays an important role in regulating the rate of insulin secretion after glucose stimulation.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.     

Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.     

A signaling role of glutamine in insulin secretion

Children with hypoglycemia due to recessive loss of function mutations of the beta-cell ATP-sensitive potassium (K(ATP)) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K(ATP) channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1-/-) mice. Even though SUR1-/- mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1-/- islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1-/- islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1-/- islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-l-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid- and glucose-stimulated insulin secretion, and that beta-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.     

The insulin-suppressive effect of resveratrol - an in vitro and in vivo phenomenon

Resveratrol, a naturally occurring phytoalexin, is known to exert numerous beneficial effects in the organism. Literature data indicate that this compound may, among other effects, play a role in prevention of diabetes and diabetic complications. Resveratrol was recently found to affect insulin secretion in vitro and to change blood insulin concentrations. These effects are, however, not fully elucidated. In the present study, 1, 10 and 100microM resveratrol incubated for 90min with pancreatic islets isolated from normal rats failed to affect basal insulin release, but substantially impaired the secretory response to physiological and maximally effective glucose. In depolarized islets exposed to resveratrol, succinate-induced insulin secretion was also diminished. The blockade of somatostatin receptors substantially enhanced insulin secretion induced by 6.7mM glucose and simultaneously suppressed the inhibitory effect of 1microM resveratrol, but at 10 and 100microM, resveratrol was still able to attenuate hormone secretion. Acetylcholine clearly increased the insulin-secretory response to 6.7mM glucose and canceled the inhibitory effect of 1microM resveratrol. However, resveratrol at concentrations 10 and 100microM strongly decreased insulin secretion. The direct activation of protein kinase C totally suppressed the inhibitory influence of 1 and 10microM resveratrol on hormone secretion. However, activation of this enzyme appeared to be insufficient to cancel the insulin-suppressive effect of 100microM resveratrol. These data indicate that resveratrol-induced inhibition of insulin secretion may be partially mitigated by suppression of somatostatin action, activation of protein kinase C or the presence of acetylcholine. The in vivo experiment revealed that resveratrol, administered to normal rats at the dose 50mg/kg body weight, diminished blood insulin concentrations at 30min, without concomitant changes in glycemia. These observations point to the direct insulin-suppressive action of resveratrol in the rat.     

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

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

Hyaluronidase-induced inhibition of the insulinotropic action of sulfonylureas, leucine, and glucagon in rodent isolated pancreatic islets.

Exposure of hamster pancreatic islets to hyaluronidase during isolation by means of collagenase inhibits the insulinotropic action of several chemically different sulfonylureas, leucine, and glucagon without affecting glucose-stimulated insulin secretion. This inhibition is reversible for tolbutamide and leucine but irreversible for glucagon. Hyaluronidase inhibits reversibly the insulinotropic action of tolbutamide without affecting that of glucose also in mouse and rat isolated pancreatic islets . These findings suggest the existence of functionally related pancreatic beta cell receptors for tolbutamide and leucine different from those for glucose and glucagon and illustrate the potential usefulness of hyaluronidase as an enzymatic probe applicable toward investigating the cellular mechanism of action of key insulinotropic agents.