Preserved initiatory and potentiatory effect of α-ketoisocaproate on insulin release in islets of glucose intolerant rats

Summary Insulin responses to glucose and non-glucose secretagogues were studied in short-term cultured pancreatic islets and perfused pancreata of the glucose intolerant F₁ hybrid rats of spontaneously diabetic Goto-Kakizaki and control Wistar rats. After culture at 5.5 mmol/l glucose, hybrid islet responses to 11.1, 16.7 and 27.0 mmol/l glucose were between 60 and 40 % of control islet responses. A combination of 1 mmol/l isobutylmethylxanthine and 16.7 mmol/l glucose induced a pronounced insulin release, which was of similar magnitude in hybrid and control rat islets. This response was not further augmented by addition of glibenclamide and arginine. The slope of potentiation of arginine (10 mmol/l)-stimulated insulin secretion by glucose (5.5–16.7 mmol/l) was greatly impaired in hybrid islets. In contrast to glucose, α-ketoisocaproate (KIC), which is metabolized in Krebs cycle, dose-dependently stimulated insulin secretion to similar levels in hybrid and control islets, cultured at 5.5 mmol/l glucose. Also in hybrid islets depolarized by potassium chloride (30 mmol/l) and with adenosine triphosphate-sensitive K⁺-channels kept open by diazoxide, insulin responses to glucose were greatly impaired but intact to KIC. Furthermore, KIC potentiated normally the insulin response to arginine in hybrid islets. In the isolated perfused pancreas, KIC induced similar insulin responses in hybrid rats and control rats. The potentiating effect by 5.5 mmol/l glucose on the KIC-stimulated insulin responses was, however, greatly reduced in isolated islets and absent in the perfused pancreata of hybrid rats. Taken together, these findings suggest an intact capacity for insulin release, although the initiating and potentiating effect by glucose on insulin release are defective in the Goto-Kakizaki-hybrid rats. An abnormal beta-cell glucose metabolism proximal to the Krebs cycle is likely to account for the impairment of insulin release. [Diabetologia (1998) 41: 1368–1373] Received: 23 March 1998 and in revised form: 23 April 1998     

Clinical measures of islet function: usefulness to characterize defects in diabetes

In healthy individuals, the ability of the pancreatic islets to sense and respond appropriately to changes in plasma glucose levels maintains plasma glucose levels within a narrow range despite broad fluctuations in nutrient intake and variable "demand" for insulin imposed by changes in insulin sensitivity. This ability of the pancreatic islets is lost in type 2 diabetes (T2DM). For studies on the pathophysiology of T2DM, methods for analyzing islet function are therefore required. Many methods of varying degrees of complexity have been developed and used to measure pancreatic beta-cell function in humans and to characterize the defects existing in patients with T2DM or precursors thereof (impaired fasting glucose [IFG] and impaired glucose tolerance [IGT]). Significant, although perhaps less progress has been made toward development of methods to characterize alpha-cell function. This work presents an overview of clinical measures of islet function, from simple static measures such as HOMA-beta to the more complex dynamic measures such as those utilizing stepped hyperglycemic clamps and acute administration of arginine to obtain more detailed information regarding the interaction of glucose and non-glucose secretagogues. We emphazise the need for accurate measures of alpha-cell function, and we discuss the strengths and limitations of the various methods, highlighting the many aspects of both alpha- and beta-cell function that become impaired during development of T2DM.     

Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells.

The high-capacity glucose transporter known as GLUT-2 and the glucose phosphorylating enzyme glucokinase are thought to be key components of the "glucose-sensing apparatus" that regulates insulin release from the beta cells of the islets of Langerhans in response to changes in external glucose concentration. AtT-20ins cells are derived from anterior pituitary cells and are like beta cells in that they express glucokinase and have been engineered to secrete correctly processed insulin in response to analogs of cAMP, but, unlike beta cells, they fail to respond to glucose and lack GLUT-2 expression. Herein we demonstrate that stable transfection of AtT-20ins cells with the GLUT-2 cDNA confers glucose-stimulated insulin secretion and glucose regulation of insulin biosynthesis and also results in glucose potentiation of the secretory response to non-glucose secretagogues. This work represents a first step toward creation of a genetically engineered "artificial beta cell."     

Perifusion of monolayer-cultured B cells of the infant rat: a comparative study of the effects of 2-deoxy-2-fluoroglucose and iodoacetic acid

Cell culture techniques for monolayer islets of 3-week-old rat pancreases and the responsiveness of B cells are described. In this procedure, whole pancreatic tissues from 3-week-old rats were enzymatically dispersed and then cultured in a medium with 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose or with 5.5 mM glucose following a 3-day exposure to a medium with 5.5mM glucose plus 5 microM iodoacetic acid. The use of 2-deoxy-2-fluoroglucose or iodoacetic acid allowed a selective deletion of fibroblasts, yielding large clusters that consisted mostly of islet cells. The immunocytochemical evaluation of the islet cells in these cultures showed that approximately 70% are B-cells, 20% A-cells, and 10% D-cells. On day 0, the response to 16.7 mM glucose included only a small rise in insulin secreted during the first and the second phase, and the response to 10 mM of leucine or 2-ketoisocaproate was monophasic. After being cultured for 7 days, all three secretagogues markedly stimulated insulin secretion by B cells cultured in both media, resulting in an enhancement of the biphasic pattern. However, quantitative relationships differed. Thus, the total response from B cells in 2-deoxy-2-fluoroglucose during a 30-min stimulation with glucose and leucine was significantly higher (1.6- and 1.9-fold respectively) than that from B cells in 5.5 mM glucose, although there was no significant difference in insulin secretion evoked by 2-ketoisocaproate. Furthermore, in the former B cells, the amount of insulin secreted during the second phase was 84-94% of the total insulin secretion, and in the latter it was 66-76%. Addition of 1 mM 3-isobutyl-1-methylxanthine and 10 microM forskolin resulted in a significant increase in insulin secretion by B cells in 2-deoxy-2-fluoroglucose, whereas there was no difference in the increase of insulin secretion induced by 16.7 mM glucose and 200 nM 12-o-tetradecanoyl phorbol-13-acetate. In monolayer cultures that had been maintained in both media for 15 days, the second phase of insulin secretion due to the secretagogues was slightly decreased, but the biphasicity in the response was well preserved. In conclusion, the present results suggest that B cells of 3-week-old rats may be still immature, and that the medium with 2-deoxy-2-fluoroglucose is beneficial to the continued maturation of the B-cell function in vitro.     

Monolayer cultures of pancreatic islet cells from adult rats: effect of 2-deoxyglucose

Techniques for the monolayer culture of pancreatic islet cells from adult rats and the responsiveness of B cells are described. Whole pancreatic tissue was enzymatically dispersed and then cultured for 30 days in tissue culture medium 199 containing 5.5 mmol glucose/l, with or without 1 mmol 2-deoxyglucose/l. In the absence of 2-deoxyglucose, the responsiveness of B cells diminished to almost zero by day 15 and islets degenerated. In contrast, addition of 2-deoxyglucose to the medium resulted in a selective degeneration of fibroblasts, yielding monolayers that consisted mostly of islet cells. In this stationary system in which monolayers of islet cells were maintained in medium with 2-deoxyglucose, insulin secretion from B cells on days 15 and 30 increased in a dose-dependent fashion in response to increasing concentrations of glucose, leucine and 2-ketoisocaproate. Similarly, when exposed to 16.7 mmol glucose/l, perifused B cells showed a biphasic pattern of insulin secretion on day 15. Addition of 10 mumol forskolin/l and 200 nmol 12-O-tetradecanoyl phorbol-13-acetate/l remarkably enhanced this response. Likewise, the response to 10 mmol leucine/l or 10 mmol 2-ketoisocaproate/l was biphasic. These results suggest that these monolayer cultures retain the functional properties of the adult rat pancreas, and may be useful not only as a model for the in-vitro study of B cell function, but also for implantation.     

Metabolic adaption of pancreatic islet tissue in aging rats

A previous report indicating reduced glucose metabolism in pancreatic islets isolated from old as compared with young rats was reinvestigated. With a modified islet isolation procedure it was found that islets from 12- to 18-month-old rats had increased glucose use, elevated glucokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase when compared with islets from 2-month-old controls. Glucose-induced insulin release in vitro of islets from the older rats was also improved by the more careful method of islet isolation but did not achieve rates observed with islets from young rats. The data suggest an age-related activation of pancreatic islet cell metabolism, possibly in response to overstimulation by increased peripheral insulin resistance, characteristic of older obese rats.     

Reduced expression of the liver/beta-cell glucose transporter isoform in glucose-insensitive pancreatic beta cells of diabetic rats.

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.     

The antilipolytic agent 3,5-dimethylpyrazole inhibits insulin release in response to both nutrient secretagogues and cyclic adenosine monophosphate agonists in isolated rat islets.

This study intended to test the hypothesis that intracellular lipolysis in the pancreatic beta cells is implicated in the regulation of insulin secretion stimulated by nutrient secretagogues or cyclic adenosine monophosphate (cAMP) agonists. Indeed, although lipid signaling molecules were repeatedly reported to influence beta-cell function, the contribution of intracellular triglycerides to the generation of these molecules has remained elusive. Thus, we have studied insulin secretion of isolated rat pancreatic islets in response to various secretagogues in the presence or absence of 3,5-dimethylpyrazole (DMP), a water-soluble and highly effective antilipolytic agent, as previously shown in vivo. In vitro exposure of islets to DMP resulted in an inhibition (by approximately 50%) of the insulin release stimulated not only by high glucose, but also by another nutrient secretagogue, 2-ketoisocaproate, as well as the cAMP agonists 3-isobutyl-1-methylxanthine and glucagon. The inhibitory effect of DMP, which was not due to alteration of islet glucose oxidation, could be reversed upon addition of sn-1,2-dioctanoylglycerol, a synthetic diglyceride, which activates protein kinase C. The results provide direct pharmacologic evidence supporting the concept that endogenous beta-cell lipolysis plays an important role in the generation of lipid signaling molecules involved in the control of insulin secretion in response to both fuel stimuli and cAMP agonists.     

Fat-induced changes in mouse pancreatic islet insulin secretion,insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with -ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.     

Forskolin-induced activation of adenylate cyclase, cyclic adenosine monophosphate production and insulin release in rat pancreatic islets

Forskolin activated adenylate cyclase in rat islet homogenates and stimulated cAMP production in intact islets incubated in the absence or presence of either D-glucose or Ca2+. Forskolin failed to affect D-[U-14C]glucose oxidation, glucose-stimulated net 45Ca uptake, or basal insulin release, but enhanced insulin secretion evoked by either nutrients (D-glucose, 2-ketoisocaproate, L-leucine alone or in combination with L-glutamine), or nonnutrient secretagogues (12-O-tetradecanoylphorbol-13-acetate, Ba2+ alone or in combination with theophylline). Forskolin stimulated insulin release from islets incubated in the presence of glucose but in the absence of Ca2+. These findings confirm that a marked increase in cAMP production is not sufficient to cause sustained insulin release. They also suggest that the enhancing action of endogenous cAMP upon insulin release does not depend on a facilitation of Ca2+ influx into islet cells.     

Effects of thioacetamide on pancreatic islet B-cell function

Thioacetamide (0.01–1.3 mM) fails to exert any significant immediate effect upon insulin release from rat isolated islets. However, when administered (4 μmol/g body wt) intraperitoneally 24 h before sacrifice, it reduced food intake and body weight and affected the secretory response of isolated islets to several secretagogues, despite unaltered insulin content of such islets. This coincided with a decrease in d-[U-¹⁴C]glucose oxidation, total islet calcium content and the ionized calcium content of secretory granules in islet B-cells, and changes in both ¹³³Ba and ⁴⁵Ca net uptake. Likewise, in islets prepared from thioacetamide-injected rats and prelabeled with ⁴⁵Ca before perifusion, the cationic and insulin secretory responses to d-glucose or gliclazide, but not to the association of Ba²⁺ and theophylline in the absence of extracellular Ca²⁺, often differed from that otherwise found in islets prepared from control rats. These findings are interpreted as indicative of an impaired capacity of Ca²⁺ sequestration by intracellular organelles in the islet B-cells of thioacetamide-treated rats.     

Impairment of the priming effect of glucose on insulin secretion from isolated islets of aging rats

The time-dependent potentiation (TDP) of insulin release or priming effect exerted by glucose was evaluated in the islets of Langerhans of mature and old rats. Islets isolated from 12-and 26-month-old male Sprague-Dawley rats and incubated for two consecutive 60-min periods in the presence of various stimulating agents were unable to enhance their insulin responsiveness significantly during the second incubation period and showed other abnormalities in their sensitivity to secretagogues compared with islets from 3-month-old animals. The priming action of glucose plus arginine or isobutylmethylxanthine (IBMX) was not observed in islets from 12-month-old rats, but surprisingly, islets from senescent rats showed a restoration of the beta-cell memory in the presence of IBMX. Interestingly, the islets isolated from 2-month-old animals previously exposed to an intravenous glucose load in vivo released approximately twice as much insulin as the islets taken from fed rats not subjected to the load. This potentiation exerted by the intravenous glucose administration was reduced but not abolished in the islets of glucose-intolerant, 12-month-old rats. In conclusion, the glucose TDP of insulin secretion is impaired in islets of mature and old rats, confirming an early loss of sensitivity of beta-cells to secretagogues during aging.     

Defective regulation of glucokinase in rat pancreatic islet cell tumours

The role of glucokinase in the regulation of insulin secretion was examined in normal rat pancreatic islets and in chemically- and radiation-induced rat pancreatic B-cell tumours which show an impaired insulin secretory response to glucose. In normal rats glucokinase activity in cytoplasmic fractions of pancreatic islets was decreased with the duration of fasting and increased by refeeding or insulin administration. This observation is consistent with the induction of glucokinase by insulin. Hexokinase activity was only slightly reduced during fasting. Glucokinase activity decreased in cytoplasmic fractions of streptozotocin-nicotinamide-induced rat pancreatic islet cell tumours. Glucokinase activity contributed about 75% to the total glucose phosphorylation capacity in cytoplasmic fractions of normal pancreatic islets and of small (less than 1 mg) streptozotocin-nicotinamide-tumours. This proportion decreased to about 20% in the large streptozotocin-nicotinamide tumours. Glucokinase activity in cytoplasmic fractions of transplantable radiation-induced NEDH (New England Deaconess Hospital) rat B-cell tumours was seven times lower than in normal pancreatic islets and contributed only 15% to the total glucose phosphorylation capacity. In contrast, hexokinase activity of the NEDH tumour B-cells was 2.5 times higher than normal. Decreased glucokinase activity in the chemically- and radiation-induced tumour B-cells appears to result from a loss of the ability of insulin to induce this enzyme and may explain the lack of insulin secretory responsiveness of these tumour B-cells.     

The role of reduced glucose transporter content and glucose metabolism in the immature secretory responses of fetal rat pancreatic islets

Summary Isolated fetal islets show an immature or poor secretory response to nutrient secretagogues which may result from impaired mitochondrial oxidative processes. Insulin secretion, glucose metabolism and detection of metabolic enzymes by radiolabelling and immunoprecipitation were compared in islets isolated from neonatal (aged 5 days) and fetal rats (at 20 days gestation). The insulin secretory dynamics of fetal islets were abnormal in response to stimulation by glucose (10 mmol/l); a rapid release of insulin reaching a maximum 6 min after stimulation was observed with no rising second phase release. However, when the data were expressed as percentage of islet insulin content released, fetal islets released significantly more insulin than neonatal islets in response to glucose (4.86±0.45 % vs 1.81±0.62 %, p <0.01) or 100 nmol/l glibenclamide (2.49±0.17 % vs 0.25±0.06 %, p < 0.001). Fetal islets however, failed to release insulin in response to stimulation by glyceraldehyde (10 mmol/l) unlike neonatal islets. Both glucose utilisation (as measured by the formation of [³H] H₂O from 5-[³H] glucose) and glucose oxidation (as measured by the formation of [¹⁴C] CO₂ from U-[¹⁴C] glucose) did not increase significantly in response to increasing the medium glucose concentration to 10 mmol/l whereas in neonatal islets, glucose utilisation and glucose oxidation were significantly increased 2.5- and 2.7-fold, respectively. When islets were incubated with both radiolabelled glucoses simultaneously, the rate of glucose oxidation was shown to be directly proportional to the rate of glucose utilisation. The relationship between glucose utilisation and glucose oxidation was similar in fetal and neonatal islets. Finally, in experiments to detect and semiquantify metabolic enzymes, the level of GLUT-2 transporter protein was significantly reduced by 50 % (p <0.02) whereas the levels of pyruvate dehydrogenase peptides were similar in fetal and neonatal islets. In conclusion, these data do not support the hypothesis that abnormal mitochondrial oxidation is responsible for the immature secretory responses to nutrient secretagogues found in fetal islets but rather that step(s) earlier in the glycolytic pathway are important for development of normal secretory function. [Diabetologia (1994) 37: 134–140] Received: 1 June 1993 and in revised form: 16 August 1993     

Pancreatic islets of obese hyperglycemic mice (ob/ob).

The development of the obesity-associated hyperglycemic syndrome in ob/ob mice, genetically determined, was observed over time by a combined functional and structural study of pancreatic islets. Islet areas increased with advancing age in ob/ob mice from 2 times at 1 month to 30 times at 6 months of age the size of lean mouse islets. Islet areas apparently increased more than pancreatic insulin content in ob/ob mice. Glucose and insulin tolerance tests were performed to study in vivo responses to glucose and insulin, respectively, in 1-, 3-, and 6-month-old mice. With ob/ob mice, glucose tolerance tests revealed more elevation of plasma glucose than in lean mice, the lean mice revealing more elevated plasma insulin than the obese mice. In insulin tolerance tests, lean mice presented marked hypoglycemia, whereas ob/ob mice revealed slightly higher plasma glucose at 1 month of age but three to four times higher amounts than that of lean mice at 6 months of age. Thus, increasing insulin resistance in ob/ob mice older than 3 months is associated with progressively increasing islet area, which contains proportionally less insulin than that of lean mouse pancreas. The data suggest that insulin resistance in ob/ob mice progressively develops up to 6 months of age and that marked islet hyperplasia is likely in response to sustained hyperglycemia, leading to hyperinsulinemia and eventual marked obesity.     

Hyperresponse in calcium-induced insulin release from electrically permeabilized pancreatic islets of diabetic GK rats and its defective augmentation by glucose

Summary In spontaneously diabetic GK rats, insulin secretion from pancreatic beta cells in response to glucose is selectively impaired, probably due to deficient intracellular metabolism of glucose and impaired closure of K_ATP channels during glucose stimulation. By using electrically permeabilized islets of GK rats, we explored the functional modulations in exocytotic steps distal to the rise in [Ca^{2 +} ]i in the diabetic condition. At 30 nmol/l Ca^{2 +} (basal conditions) insulin release was similar between GK and non-diabetic control Wistar rats. In response to 3.0 μmol/l Ca^{2 +} (maximum stimulatory conditions), insulin release was significantly augmented in permeabilized GK islets (p < 0.01). Raising glucose concentrations from 2.8 to 16.7 mmol/l further augmented insulin release induced by 3.0 μmol/l Ca^{2 +} from permeabilized control islets(p < 0.001), but had no effect on that from permeabilized GK islets. The stimulatory effect of glucose on insulin release from permeabilized control islets was partly inhibited by 2,4-dinitrophenol, an inhibitor of mitochondrial oxidative phosphorylation (p < 0.01). The hyperresponse to Ca^{2 +} in GK islets may play a physiologically compensatory role on the putative functional impairment both in [Ca^{2 +} ]i rise and energy state in response to glucose in diabetic β cells, and may explain the relative preservation of insulin release induced by non-glucose depolarizing stimuli, such as arginine, from pancreatic islets in non-insulin-dependent diabetes mellitus. [Diabetologia (1995) 38: 772–778] Received: 17 September 1994 and in revised form: 29 December 1994     

Effects of glucose, leucine and adenosine on insulin release, 45Ca2+ net uptake, NADH/NAD ratios and oxygen consumption of islets isolated from fed and starved mice

In order to elucidate further the effects of starvation on islet metabolism and insulin release, pancreatic islets of mice were isolated and incubated in the presence of various nutrient secretagogues. Starvation for 60 h completely blocked the insulin release in response to either 16.7 mM glucose or 10 mM leucine. The further addition of 20 mM adenosine partly restored the insulin response. Glucose, adenosine, glucose + adenosine, glucose + leucine or leucine + adenosine all increased the NADH/NAD ratios over basal values in islets from both fed and starved mice. No effects of starvation were observed on islet NADH/NAD ratios in any of the above media, but when islets of starved animals were incubated in the absence of any metabolic substrates the NADH/NAD ratios were decreased. In the absence of exogenous substrates the respiratory rate was also lower in islets from starved animals. Respiratory stimulation evoked by either 16.7 mM glucose or 10 mM leucine + 10 mM glutamine was lower after starvation, whereas glucose + adenosine, glucose + leucine and adenosine all induced normal respiratory responses. No differences between the 45Ca2+ uptake of islets from either starved or fed mice were observed under any conditions. It is concluded that, in starvation, a dissociation between islet insulin release and metabolism (measured as NADH/NAD ratios, oxygen consumption and 45Ca2+ uptake) may exist in the presence of certain nutrient secretagogues.     

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer