Enhanced β-adrenergic receptors in the brain and pancreas during pancreatic regeneration in weanling rats

Adrenergic stimulation has an important role in the pancreatic β-cell proliferation and insulin secretion. In the present study, we have investigated how sympathetic system regulates the pancreatic regeneration by analyzing Epinephrine (EPI), Norepinephrine (NE) and β-adrenergic receptor changes in the brain as well as in the pancreas. EPI and NE showed a significant decrease in the brain regions, pancreas and plasma at 72 hrs after partial pancreatectomy. We observed an increase in the circulating insulin levels at 72 hrs. Scatchard analysis using [³H] propranolol showed a significant increase in the number of both the low affinity and high affinity β-adrenergic receptors in cerebral cortex and hypothalamus of partially pancreatectomised rats during peak DNA synthesis. The affinity of the receptors decreased significantly in the low and high affinity receptors of cerebral cortex and the high affinity hypothalamic receptors. In the brain stem, low affinity receptors were increased significantly during regeneration whereas there was no change in the high affinity receptors. The pancreatic β-adrenergic receptors were also up regulated at 72 hrs after partial pancreatectomy. In vitro studies showed that β-adrenergic receptors are positive regulators of islet cell proliferation and insulin secretion. Thus our results suggest that the β-adrenergic receptors are functionally enhanced during pancreatic regeneration, which in turn increases pancreatic β-cell proliferation and insulin secretion in weanling rats.     

Decreased 5-HT2C receptor binding in the cerebral cortex and brain stem during pancreatic regeneration in rats

The purpose of this study was to investigate the role of central 5-HT_2C receptor binding in rat model of pancreatic regeneration using 60–70% pancreatectomy. The 5-HT and 5-HT_2C receptor kinetics were studied in cerebral cortex and brain stem of sham operated, 72 h pancreatectomised and 7 days pancreatectomised rats. Scatchard analysis with [³H] mesulergine in cerebral cortex showed a significant decrease (p < 0.05) in maximal binding (B_max) without any change in K_d in 72 h pancreatectomised rats compared with sham. The decreased B_max reversed to sham level by 7 days after pancreatectomy. In brain stem, Scatchard analysis showed a significant decrease (p < 0.01) in B_max with a significant increase (p < 0.01) in K_d. Competition analysis in brain stem showed a shift in affinity towards a low affinity. These parameters were reversed to sham level by 7 days after pancreatectomy. Thus the results suggest that 5-HT through the 5-HT_2C receptor in the brain has a functional regulatory role in the pancreatic regeneration.     

Decreased α1-Adrenergic Receptor Binding in the Cerebral Cortex and Brain Stem during Pancreatic Regeneration in Rats

The purpose of this study was to investigate the role of brain α₁-adrenergic receptor binding in the rat model of pancreatic regeneration using 60–70% pancreatectomy. The α₁-adrenergic receptors kinetics was studied in the cerebral cortex and brain stem of sham operated, 72 h pancreatectomised and 7 days pancreatectomised rats. Scatchard analysis with [³H]prazosin in cerebral cortex and brain stem showed a significant decrease (P < 0.01), (P < 0.05) in maximal binding (B _max) with a significant decrease (P < 0.001), (P < 0.01) in the K _d in 72 h pancreatectomised rats compared with sham respectively. Competition analysis in cerebral cortex and brain stem showed a shift in affinity during pancreatic regeneration. The sympathetic activity was decreased as indicated by the significantly decreased norepinephrine level in the plasma (P < 0.001), cerebral cortex (P < 0.01) and brain stem (P < 0.001) of 72 h pancreatectomised rats compared to sham. Thus, from our results it is suggested that the central α₁-adrenergic receptors have a functional role in the pancreatic regeneration mediated through the sympathetic pathway.     

Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.

Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.     

Regulation of insulin and glucagon secretion from rat pancreatic islets in vitro by somatostatin analogues

Somatostatin is an inhibitor of hormone secretion through specific receptors (sst1-5). The aim of this study was to investigate the putative regulatory role of somatostatin analogues on the secretion of insulin and glucagon by rat pancreatic islets. After 48 h exposure only the non-selective agonists (somatostatin, octreotide and SOM-230) inhibited insulin accumulation. The inhibition of insulin secretion was accompanied by increased islet insulin contents. None of the analogues showed a consistent effect on the glucagon accumulation in the medium after 48 h. Since we observed a difference in the regulatory effect between the non-selective and selective analogues, combinations of selective analogues were studied. Combination of sst2+sst5 agonists inhibited the medium insulin accumulation, while combination of sst1+sst2 analogues caused a decrease in glucagon accumulation. After removal of somatostatin a rebound effect with increased insulin secretion were observed. This effect was reversed after 6 h. For SOM-230 insulin secretion continued to be suppressed even after the analogue was removed and returned to control values after 3 h. As for glucagon secretion there was an initial decline after culture with octreotide, while the other substances failed to induce any changes. In summary, non-selective somatostatin analogues or combinations of receptor selective analogues may cause inhibition of hormone secretion from rat pancreatic islets. For insulin and glucagon, combinations of sst2+sst5 and sst1+sst2, respectively may exert this effects. Thus, our data suggest that more than one sst must be involved to down-regulate islet glucagon and insulin secretion.     

Alterations in the muscarinic M1 and M3 receptor gene expression in the brain stem during pancreatic regeneration and insulin secretion in weanling rats

Muscarinic M1 and M3 receptor changes in the brain stem during pancreatic regeneration were investigated. Brain stem acetylcholine esterase activity decreased at the time of regeneration. Sympathetic activity also decreased as indicated by the norepinephrine (NE) and epinephrine (EPI) content of adrenals and also in the plasma. Muscarinic M1 and M3 receptors showed reciprocal changes in the brain stem during regeneration. Muscarinic M1 receptor number decreased at time of regeneration without any change in the affinity. High affinity M3 receptors showed an increase in the number. The affinity did not show any change. The number of low affinity receptors decreased with decreased Kd at 72 hours after partial pancreatectomy. The Kd reversed to control value with a reversal of the number of receptors to near control value. Gene expression studies also showed a similar change in the mRNA level of M1 and M3 receptors. These alterations in the muscarinic receptors regulate sympathetic activity and maintain glucose level during pancreatic regeneration. Central muscarinic M1 and M3 receptor subtypes functional balance is suggested to regulate sympathetic and parasympathetic activity, which in turn control the islet cell proliferation and glucose homeostasis.     

Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats

Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D₃ in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D₃ treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D₃ supplementation improved the behavioural deficit. In conclusion, vitamin D₃ shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D₃ modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D₃ supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D₃ and muscarinic M3 receptors in regulating insulin secretion from pancreas.     

A microperifusion system with environmental control for studying insulin secretion by pancreatic tissue.

A continuous flow reactor (perifusion system) was fabricated and tested for measuring the kinetics of insulin secretion from isolated pancreatic islets of Langerhans in response to step changes in the glucose concentration and oxygen partial pressure in the perfusate flowing around the islets. The system was capable of making rapid changes in perfusate glucose concentration and pO2, had rapid dynamic response for measuring the change in insulin secretion rate as a result of these changes in perfusate, and was suitable for studying very small volumes of tissue. Initial experiments with this system demonstrated that (1) the response of isolated rat islets to glucose stimulation was very fast, with the first phase peak occurring in as little as about 10 s, (2) bulk perfusate oxygen partial pressure levels of 30 mmHg or less reduced the second-phase insulin secretion rate in graded fashion, (3) the reduction in secretion rate began within 1 min following an oxygen partial pressure decrease, and (4) the reduction in secretion rate was reversible, with a burst of insulin secretion occurring during the first minute after partial pressure restoration.     

Hypothalamic adrenergic receptor changes in the metabolic syndrome of genetically obese (ob/ob) mice

The genetically, seasonally, and diet-induced obese, glucose-intolerant states in rodents, including ob/ob mice, have each been associated with elevated hypothalamic levels of norepinephrine (NE). With the use of quantitative autoradiography on brain slices of 6-wk-old obese (ob/ob) and lean mice, the adrenergic receptor populations in several hypothalamic nuclei were examined. The binding of [(125)I]iodocyanopindolol to beta(1)- and beta(2)-adrenergic receptors in ob/ob mice was significantly increased in the paraventricular hypothalamic nucleus (PVN) by 30 and 38%, in the ventromedial hypothalamus (VMH) by 23 and 72%, and in the lateral hypothalamus (LH) by 10 and 15%, respectively, relative to lean controls. The binding of [(125)I]iodo-4-hydroxyphenyl-ethyl-aminomethyl-tetralone to alpha(1)-adrenergic receptors was also significantly increased in the PVN (26%), VMH (67%), and LH (21%) of ob/ob mice. In contrast, the binding of [(125)I]paraiodoclonidine to alpha(2)-adrenergic receptors in ob/ob mice was significantly decreased in the VMH (38%) and the dorsomedial hypothalamus (17%) relative to lean controls. This decrease was evident in the alpha(2A)- but not the alpha(2BC)-receptor subtype. Scatchard analysis confirmed this decreased density of alpha(2)-receptors in ob/ob mice. Together with earlier studies, these changes in hypothalamic adrenergic receptors support a role for increased hypothalamic NE activity in the development of the metabolic syndrome of ob/ob mice.     

UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells

Background

Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets.

Methodology

Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry.

Principal Findings

Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion.

Conclusions

UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.     

Properties of the insulin receptor of rat pancreatic islet

Rat pancreatic islets have been shown to possess specific binding sites for ¹²⁵I-labeled insulin. Enzymatic and chemical modification of islets are used to reveal important structures and chemical groups for insulin binding. Pretreatment with trypsin, neuraminidase, 1-ethyl-3(3-dimethylamino)carbodiimide (a carboxyl reagent), tetranitromethane (a tyrosyl and thiol reagent), and 1,3-difluoro-4,6-dinitrobenze (modification of protein functional groups) decreased binding of insulin. This was due to the diminuation of the receptor number; in the case of trypsin-pretreatment also the receptor affinity was decreased. Inhibition of insulin binding was in each case associated with a decrease of the inhibitory effect of exogenous insulin on glucose-induced insulin secretion (not measured in the case of difluorodinitrobenzene and tetranitromethane). Phospholipase A₂ (cleavage of phospholipids) did not affect these parameters. 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) and possibly p-chloromercuribenzoate (both thiol reagents) increased the number of receptors and decreased receptor affinity, but did not influence the inhibitory effect of insulin on insulin release. It is concluded that protein functional groups, sialic acid, carboxyl and tyrosyl groups, but not phospholipids and probably not sylfhyryl groups are important for the interaction of insulin with insulin receptors of rat pancreatic islets.     

Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion.

The influence of down-regulation of protein kinase C on glucose-induced insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM) in RPMI 1640 culture medium (8.3 mM-glucose, 0.43 mM-Ca2+) abolished TPA (0.16 microM)-induced insulin secretion and led to a potentiation of phase 1 and a decrease in phase 2 of glucose-induced insulin secretion. Thus, although the total insulin release during 40 min of perfusion with glucose (16.7 mM) (45-85 min) was unaffected, the percentage released during phase 1 (45-55 min) was increased from 12.9 +/- 1.5 (4)% in controls to 35.8 +/- 3.9 (4)% in TPA-treated islets (P less than 0.01), and the percentage released during phase 2 (65-85 min) was decreased from 63.2 +/- 3.9 (4)% to 35.3 +/- 1.4 (4)% (P less than 0.005). In contrast, TPA exposure in TCM 199 medium (5.5 mM-glucose, 1.26 mM-Ca2+) caused a total abolition of both phases 1 and 2 of glucose-induced secretion. However, inclusion of the alpha 2-adrenergic agonists adrenaline (10 microM) or clonidine (10 microM), or lowering of the Ca2+ concentration in TCM 199 during down-regulation, preserved and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1 and inhibited phase 2 of carbamoylcholine (100 microM)-induced insulin secretion at 3.3 mM-glucose, and abolished the potentiating effect of carbamoylcholine (100 microM) at 16.7 mM-glucose. These results substantiate a role for protein kinase C in insulin secretion, and suggest that protein kinase C inhibits phase 1 and stimulates phase 2 of both glucose-induced and carbamoylcholine-induced insulin secretion.     

Leptin suppresses basal insulin secretion from rat pancreatic islets

The effects of leptin on insulin secretion from pancreatic islets of Sprague–Dawley rats were examined in vitro. In a basal glucose medium (5.5 mM), insulin secretion from isolated islets was significantly decreased after addition of a recombinant leptin (80 nM) (3.20±0.14 nmol/10 islets/h) compared with that before the addition (4.41±0.30 nmol/10 islets/h). Although significant leptin suppression of insulin secretion was not observed under a glucose-stimulated (11.1 mM) condition, these results suggest that a negative feedback system may exist between leptin and insulin, which increases the production of leptin from adipose tissues.     

PPAR-gamma overexpression selectively suppresses insulin secretory capacity in isolated pancreatic islets through induction of UCP-2 protein

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions, but its physiological role in pancreatic islet cells remains to be investigated. In this study, we confirmed the presence of PPAR-gamma in rat isolated islets and examined its role on insulin and glucagon secretion by using PPAR-gamma-overexpressed islets. PPAR-gamma overexpression significantly suppressed insulin secretion induced by stimulatory concentration of glucose (p<0.05). In addition, insulin secretion evoked by high potassium depolarization also was significantly decreased from PPAR-gamma-overexpressed islets (p<0.05). On the other hand, no significant change in glucagon release was observed after high potassium depolarization between PPAR-gamma-overexpressed and control islets. Insulin and glucagon content in islets was not statistically different between the two groups. In addition, the expression of uncoupling protein-2 (UCP-2) was found to be induced in PPAR-gamma-overexpressed islets. This result clearly indicates that the deteriorative effect of PPAR-gamma overexpression on the secretory machinery is selective for pancreatic beta-cells. And it is possible that its site of action can be located in the energy-consuming exocytotic process of insulin secretory granules, and that the reduction of ATP production through increased UCP-2 reduces insulin exocytosis.     

Insulinotropic effect of the tumor promoter teleocidin in isolated pancreatic islets

A tumor promoter teleocidin induced insulin secretion from isolated pancreatic islets in a concentration-dependent manner. The teleocidin-induced secretion was inhibited by p-bromophenacyl bromide, nordihydroguaiaretic acid, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone, but not by indomethacin. Insulinotropic concentrations of teleocidin stimulated 6-keto-prostaglandin F1 alpha release from pancreatic islets. These results suggest that phospholipase A2 activation and lipoxygenase product(s) are involved in the mechanism of teleocidin-induced insulin secretion.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

Protein kinase Cdelta plays a non-redundant role in insulin secretion in pancreatic beta cells

Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.     

Glucose-regulated glucagon secretion requires insulin receptor expression in pancreatic alpha-cells

The insulin receptor (IR) and its signaling appear to be essential for insulin secretion from pancreatic beta-cells. However, much less is known about the role of the IR in alpha-cells. To assess the role of the IR in glucagon and insulin secretion, we engineered adeno-viruses for high efficiency small interference RNA (siRNA)-IR expression in isolated mouse pancreatic islets and lentiviruses for siRNA-IR expression in pancreatic alpha- and beta-cell lines (alpha-TC6 and MIN6) with specific, long term stable IR knockdown. Western blot analysis showed that these strategies resulted in 60-80% reduction of IR protein in islets and alpha- and beta-cell lines. Cell growth was reduced by 35-50% in alpha-TC and MIN6 cells stably expressing siRNA-IR, respectively. Importantly, glucagon secretion, in response to glucose (25 to 2.8 mm), was completely abolished in islets expressing siRNA-IR, whereas secretion increased 1.7-fold in islets expressing control siRNA. In contrast, there was no difference in glucose-stimulated insulin secretion when comparing siRNA-IR and siRNA control, with both groups showing a 1.7-fold increase. Islet glucagon and insulin content were also unaffected by IR knockdown. To further explore the role of the IR, siRNA-IR was stably expressed in pancreatic cell lines, which dramatically suppressed glucose-regulated glucagon secretion in alpha-TC6 cells (3.4-fold) but did not affect GSIS in MIN6 cells. Defects in siRNA-IR-expressing alpha-cells were associated with an alteration in the activity of Akt and p70S6K where insulin-induced phosphorylation of protein kinase B/AKt was greatly reduced while p70S6K activation was enhanced, suggesting that the related pathways play important roles in alpha cell function. This study provides direct evidence that appropriate expression of the IR in alpha-cells is required for glucose-dependent glucagon secretion.     

Imidazoline NNC77-0074 stimulates Ca2+-evoked exocytosis in INS-1E cells by a phospholipase A2-dependent mechanism

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.