Modulation of cyclic nucleotides and cyclic nucleotide phosphodiesterases in pancreatic islet beta-cells and intestinal L-cells as targets for treating diabetes mellitus

Cyclic 3'5'-AMP (cAMP) is an important physiological amplifier of glucose-induced insulin secretion by the pancreatic islet beta-cell. In the beta-cell, cAMP is formed by the activity of adenylyl cyclase, especially in response to the incretin hormones glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide. cAMP may also play a similar role in regulating GLP-1 secretion from intestinal L-cells. cAMP influences many steps involved in glucose-induced insulin secretion and may be important in regulating pancreatic islet beta-cell differentiation, growth and survival. cAMP itself is rapidly degraded in the pancreatic islet beta-cell by cyclic nucleotide phosphodiesterase enzymes. This review will discuss the possibility of targeting cAMP mechanisms in the treatment of type 2 diabetes mellitus, in which insulin release in response to glucose is impaired.     

Role of CYP2E1 in ketone-stimulated insulin release in pancreatic B-cells

The role of CYP2E1 in ketone-stimulated insulin release was investigated using isolated pancreatic islets of Langerhans and two mammalian insulin secreting pancreatic beta-cell lines engineered to stably express human CYP2E1 (designated BRIN BD11h2E1 and INS-1h2E1). Isolated rat pancreatic islets were shown to express the CYP2E1 isoform which was inducible by pretreatment of animals with acetone. The cDNA encoded CYP2E1 was expressed and inducible in the engineered cells as shown by Western blotting. The transfected protein was enzymatically active in the heterologous cells as determined by p-nitrophenol hydroxylation rates (0.176 +/- 0.08 vs. 0.341 +/- 0.08 nmol/min/mg microsomal protein in BRIN BD11 control cells and BRIN BD11h2E1 cells respectively, P < 0.001; 0.204 +/- 0.03 vs. 0.633 +/- 0.102 nmol/min/mg microsomal protein in INS-1 and INS-1h2E1, respectively, P < 0.001). Cultivation of CYP2E1 expressing BRIN BD11h2E1 and INS-1h2E1 cells in 40 mM ethanol increased the rate of p-nitrophenol hydroxylation (0.968 +/- 0.09 nmol/min/mg microsomal protein, P < 0.001 and 0.846 +/- 0.103 nmol/min/mg microsomal protein, P < 0.001, respectively) providing further evidence that the heterologous protein is inducible. Cultivation of control cells with ethanol had no observable effect (0.186 +/- 0.05 and 0.195 +/- 0.03 in BRIN BD11 and INS-1, respectively). These cell lines also express NADPH-cytochrome P450 reductase protein which was enzymatically active (0.632 +/- 0.023 in parental BRIN BD11 vs. 0.657 +/- 0.066 without ethanol and 0.824 +/- 0.014 nmol/min/mg microsomal protein with ethanol in BRIN BD11h2E1, P < 0.05; and 1.568 +/- 0.118 in parental INS-1 vs. 1.607 +/- 0.093 without ethanol and 1.805 +/- 0.066 nmol/min/mg microsomal protein with ethanol in INS-1h2E1, P < 0.05) thereby providing a functional cytochrome P450 system. The insulin secretory response of control cell lines and islets was similar to cell lines and islets which had been chemically pretreated, to induce CYP2E1 expression, in response to known nutrient secretagogues. However, insulin output was significantly higher in pretreated islets (1.3-fold, P < 0.05) and CYP2E1 expressing cell lines (BRIN BD11h2E1 2.3-fold, P < 0.001; INS1-1h2E1 1.6-fold, P < 0.001) when stimulated with the ketone 3-hydroxybutyrate than control islets and parental cell lines respectively. Similar acute exposure to acetoacetate enhanced insulin secretion by 1.3-fold (P < 0.05) in pretreated islets, 2.6-fold (P < 0.001) in ethanol pretreated BRIN BD11h2E1 and 1.4-fold (P < 0.001) in ethanol pretreated INS-1h2E1 cells compared to the respective control islets or ethanol pretreated control parental cells. Therefore, these studies highlight a possible role for CYP2E1 in pancreatic cell dysfunction.     

罗格列酮通过与IRS—2相关的磷脂酰肌醇3激酶途径逆转慢性高游离脂肪酸引起的胰岛素分泌

目的:研究罗格列酮逆转由慢性高浓度游离脂肪酸引起的胰岛素分泌的效果并探讨介导其作用的可能信号转导机制。方法:分离纯化的SD大鼠胰岛细胞用游离脂肪酸2mmol/L或/和加用罗格列酮(0.05-10μmol/L)培养。胰岛素释放功能采用放免法测定,胰岛素受体底物-2(IRS-2)蛋白的表达水平以及IRS-2与磷脂酰肌醇3激酶(PI 3K)的p85亚单位的相关作用通过免疫沉淀和蛋白质印迹分析法检测。结果:与对照组比较,对胰岛β细胞高浓度游离脂肪酸的慢性温育显著增加了基础胰岛素分泌而显著降低了葡萄糖刺激的胰岛素分泌(P<0.01),IRS-2蛋白的表达水平降低了65％(P<0.01),IRS-2与p85的相关作用降低了73％(P<0.01)。当加入罗格列酮继续培养后,基础和葡萄糖刺激的胰岛素分泌均恢复到接近对照水平(P<0.01,P<0.05),IRS-2蛋白的表达水平增加了2.6倍(P<0.01),IRS-2与p85的相关作用增加了2.7倍(P<0.01)。PI 3K抑制物wortmannin 100 nmol/L抑制了罗格列酮逆转胰岛素分泌的作用。结论:罗格列酮逆转高浓度游离脂肪酸引起的胰岛素分泌改变,可能是通过与IRS-2相关的磷脂酰肌醇3激酶途径所介导。     

L-赖氨酸对克隆的胰岛β细胞株INS-1E细胞的急性作用

目的:探讨L-赖氨酸刺激INS-1E细胞分泌胰岛素(INS)的作用。方法:INS-1E细胞经传代培养2d后,在Krebs-Ringer缓冲液中37℃培养箱预培养30min,再用含有不同浓度葡萄糖和不同浓度L-赖氨酸的改良Krebs-Ringer缓冲液培养60min,然后留取上清液进行胰岛素测定并用细胞蛋白含量校正胰岛素。结果:在1.1～6.7mmol/L葡萄糖范围内,随着葡萄糖浓度的增加,INS分泌量逐渐增加。L-赖氨酸在0.1～20mmol/L范围促进了葡萄糖刺激的INS-1E细胞的胰岛素分泌,随剂量增大而增强。结论:INS-1E细胞分泌胰岛素随葡萄糖浓度的增加而增加。L-赖氨酸能增加葡萄糖诱导的INS-1E细胞分泌胰岛素的作用。     

Effects of isoprenaline and glucagon on insulin secretion from pancreatic islets

Summary The effects of isoprenaline and glucagon on insulin secretion from pancreatic islets were investigated. In the presence of high concentrations of isoprenaline (10–50 mol/l), glucose-induced (20 mmol/l) insulin secretion from isolated perifused mouse islets was inhibited. This inhibition was apparently mediated by ₂-adrenoceptors, as it was antagonized by rauwolscine. At low concentrations isoprenaline (0.1 or 1 mol/l) did not affect glucose-induced (2.5; 10 or 20 mmol/l) insulin secretion from perifused mouse or rat islets, even if ₂-adrenoceptors were blocked by rauwolscine. A stimulatory effect of isoprenaline on insulin secretion was also not observed in the perfused rat pancreas. However, when incubated mouse islets were exposed to glucose (10 mmol/l), insulin secretion was further enhanced by isoprenaline (0.5 mol/l). To elucidate the underlying mechanism, the effects of glucagon on insulin secretion were investigated, because glucagon is released from the pancreatic A-cells during stimulation with isoprenaline and is accumulated in the islets and the surrounding medium during incubations of pancreatic islets. Indeed, glucagon stimulated insulin secretion from perifused mouse islets in the presence of high glucose (10 or 15 mmol/l) concentrations but not of low glucose (5 mmol/l) concentrations. Thus it is concluded that direct -adrenergic stimulation of pancreatic B-cells does not occur in mouse or rat pancreatic islets. Augmentation of glucose-induced insulin secretion by isoprenaline observed in incubation systems can be explained as a result of stimulation by glucagon, which is released from pancreatic A-cells by isoprenaline.Some of the results described here were obtained during medical thesis work by S. Zielmann and g. Schütte     

Inhibition of adenylyl cyclase by neuronal P2Y receptors

P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y(12). Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 - 100 microM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A(2A) or recombinant beta(2) receptors. Forskolin-dependent cyclic AMP production was reduced by ADPbetaS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, or adenosine-3'-phosphate-5'-phosphate, but attenuated by reactive blue 2, ATP(alpha)S, and 2-methylthio-AMP. RT - PCR demonstrated the expression of P2Y(2), P2Y(4), P2Y(6), and P2Y(12), but not P2Y(1), receptors in PC12 cells. In Northern blots, only P2Y(2) and P2Y(12) were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y(12) receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity.     

ADP-sensitive purinoceptors induce steroidogenesis via adenylyl cyclase activation in bovine adrenocortical fasciculata cells

1. The role of P2Y receptors in the production of cAMP and the activation of protein kinase A (PKA) was studied with respect to the regulation of the steroidogenesis in primary cultures of bovine adrenocortical fasciculata cells (BAFCs). 2. ADP and ATP stimulated cAMP production with EC(50) values of 23.7+/-6.8 microM and 40.1+/-5.5 microM, respectively. In contrast, the EC(50) of BzATP for cAMP production was 153.0+/-37.4 microM. Adenosine and AMP (0.1-1000 microM) were much less effective than ADP and ATP. 2MeSADP and UTP did not exert detectable effects. ADP (10 and 100 microM) significantly stimulated steroidogenesis; the process was blocked by an adenylyl cyclase inhibitor SQ22536 (100 microM) but not by the P2Y(1) receptor antagonist MRS2179 (100 microM). 3. Real-time imaging of the PKA activity with the dye ARII, which became less fluorescent upon phosphorylation, revealed that ADP (100 microM) immediately activated PKA. These effects could be mimicked by forskolin (100 microM) and were blocked by the PKA inhibitor H89 (50 microM). UTP (100 microM) did not activate PKA. 4. The cytoplasm harvested from morphologically and electrophysiologically identified single BAFCs contained mRNA for P2Y(2) but not for P2Y(1), P2Y(4), P2Y(11) or P2Y(12) receptors, as confirmed by single-cell RT-PCR amplification (50 cycles). 5. These results suggest an expression of an ADP-sensitive G(s)-coupled purinoceptor in BAFCs. We propose that this not yet described type of P2Y receptor might mediate the extracellular purine-activated steroidogenesis via cAMP/PKA-mediated pathways, independently from the pathways involving InsP(3) production and consequent intracellular Ca(2+) increase.     

Expression of purinergic P2Y receptor subtypes by INS-1 insulinoma beta-cells: a molecular and binding characterization

Purinergic P2Y-receptor agonists amplify glucose-induced insulin secretion from pancreatic beta-cells, thus offering new opportunities for the treatment of type 2 diabetes. However, little is known about which subtypes of purinergic P2Y receptors are expressed in these cells. The INS-1 beta-cell line is used as a model of pancreatic beta-cells, expressing most of their properties. Therefore, we investigated the expression of different molecular subtypes in this cell line by means of real time Polymerase Chain Reaction and Western blot. We also performed a characterization of the binding of a prototypic purinergic P2Y agonist, Adenosine-5'-O-(1-[(35)S]thiotriphosphate) (ATP-alpha-[(35)S]), to cell membrane homogenates. The molecular analysis evidenced the presence of five different purinergic P2Y receptor subtypes (P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12)), which were expressed at similar levels. The Western blot analysis allowed detecting corresponding proteins. The binding assay demonstrated a specific ATP-alpha-[(35)S] interaction on high (40%) and low (60%) affinity components. The analysis of ATP-alpha-[(35)S] pharmacological profile on both sites permitted to classify the high affinity binding site as representative of the purinergic P2Y(1) receptor subtype and the low affinity binding site of the P2Y(4) and/or P2Y(6) receptor subtypes. ATP-alpha-S and Adenosine-5'-O-(2-thiodiphosphate) (ADP-beta-S) exhibited opposite selectivity on high and low affinity binding sites.Although purinergic P2Y(1) receptor, or a P2Y(1)-like subtype, has been generally considered as that implicated in the modulation of glucose-induced insulin release, the present data show that the beta-cell expresses a complex profile of purinergic P2Y receptor subtypes, the functional implication of which remains to be fully elucidated.     

Regulation of responsiveness at D2 dopamine receptors by receptor desensitization and adenylyl cyclase sensitization.

The regulation of cellular responsiveness to dopamine via the D2 dopamine receptor was investigated in mouse fibroblast Ltk-cells stably expressing the rat D2-short receptor [Nature (Lond.) 336:783-787 (1988)]. Dopamine inhibited forskolin-stimulated cAMP levels in these cells (half-maximal inhibition at 3.9 +/- 1.1 nM), and the inhibition by dopamine was blocked by D2 antagonists and was pertussis toxin sensitive. Treatment of these cells with the D2 agonist quinpirole (1 microM) resulted in desensitization of dopaminergic inhibition of forskolin-stimulated cAMP accumulation, with a approximately 4-fold decrease in the potency of dopamine after 1 hr of treatment. No significant changes in total cellular D2 receptor concentrations were observed, even after prolonged agonist treatment. At longer time points, basal and forskolin-stimulated cellular cAMP levels were increased in treated cells. The effect of D2 agonist treatment on membrane adenylyl cyclase (EC 4.6.1.1) activity was examined. Basal and forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were increased by quinpirole treatment for 24 hr. This sensitization of adenylyl cyclase was blocked by the presence of a D2 antagonist. Pertussis toxin pretreatment blocked the sensitization of adenylyl cyclase by quinpirole, although pertussis toxin also caused increased adenylyl cyclase activity on its own. Sensitization was not dependent upon dopaminergic inhibition of intracellular cAMP levels, because quinpirole treatment in the presence of membrane-permeable cAMP analogs or 3-isobutyl-1-methylxanthine (an inhibitor of cAMP phosphodiesterase) resulted in greater sensitization of adenylyl cyclase activity than quinpirole treatment alone. These results suggest that, in this model system, responsiveness to dopamine via the D2 receptor is regulated by both desensitization of receptor function and sensitization of the stimulatory adenylyl cyclase pathway.     

棕榈酸对小鼠胰岛和INS-1E细胞作用的研究

目的:探讨高脂对INS-1E细胞和小鼠胰岛的慢性作用.方法:雌性NMRI小鼠6～10周龄,苯巴比妥腹腔麻醉,常规开腹,取小鼠胰腺组织,应用胶原酶技术消化胰腺分离胰岛.传代培养的INS-1E细胞和分离的小鼠胰岛分别在含与不含棕榈酸的RPMI1640中培养72 h,然后在含3.3、16.7 mmol/L葡萄糖的Krebs-Ringer缓冲液中培养60min,留取上清液行胰岛素测定.INS-1E细胞在含不同浓度的棕榈酸的RPMI1640中培养72 h,提取其总RNA,合成相应的cDNA,再行RT-PCR检测胰十二指肠同源异形盒1(Pdx1),胰岛素1、胰岛素2和葡萄糖转运子2的基因表达.结果:高脂培养后INS-1E细胞和小鼠胰岛的基础胰岛素分泌增加,糖刺激的胰岛素分泌减少,INS-1E细胞的胰岛素1,胰岛素2和葡萄糖转运子2的mRNA水平下降.结论:高脂显示了对胰岛B细胞的慢性毒性作用.     

Resveratrol inhibits insulin secretion from rat pancreatic islets

Resveratrol is a naturally occurring phytoalexin exerting cardioprotective, anticancer and antioxidant action. The most recent investigations have demonstrated that this compound plays a beneficial role alleviating some diabetic complications. However, resveratrols' influence on the endocrine function of the pancreas is unknown. The objective of the present study was to determine whether resveratrol affects insulin secretion from freshly isolated rat pancreatic islets. Incubations of pancreatic islets with resveratrol (1-100 microM, 90 min) revealed that the release of insulin induced by 6.6 and 16.6 mM glucose was substantially restricted by this compound in a concentration-dependent manner. This effect was not permanent and disappeared after resveratrol withdrawal from the buffer. However, the proper hormone secretion was not restored when glucose was replaced by other secretagogues - leucine with glutamine - indicating that disturbances other than the inhibition of glucose transport and glycolysis were responsible for the resveratrol-evoked reduction in insulin secretion. Glucose-induced insulin release tested in the presence of the sulfonylurea glibenclamide was also found to be reduced by resveratrol. Moreover, the activation of adenylyl cyclase by forskolin did not restrict the inhibitory effect of resveratrol on glucose-induced insulin release. In contrast, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, suppressed completely the inhibitory effect of 1 and 10 microM resveratrol on insulin release. However, this compound at the highest concentration tested diminished its secretion even in the presence of PMA. The perifusion studies revealed that the depression of insulin release caused by resveratrol began a few minutes after its addition to the medium. Results obtained in the present investigations demonstrate that resveratrol is a compound exerting a clear-cut, but reversible inhibitory effect on insulin secretion from isolated pancreatic islets.     

Involvement of Raf-1 in chronic delta-opioid receptor agonist-mediated adenylyl cyclase superactivation

Chronic delta-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic delta-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 microM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% (n = 6, P < 0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.     

Therapeutic effects of berberine in impaired glucose tolerance rats and its influence on insulin secretion

AIM: To explore the anti-diabetic effects of berberine and its influence on insulin secretion. METHODS: Impaired glucose tolerance rats induced by iv injection of streptozotocin 30 mg/kgwere treated with berberine 187.5 and 562.5 mg/kg while fed with high fat laboratory chow. After rats were treated for 4 weeks, oral glucose tolerance was determined, and for 8 weeks, the fasting blood glucose, insulin, lipid series were determined. In insulin secretion experiments, berberine 93.75, 187…     

不同浓度的葡萄糖对INS-1细胞cAMP生成与胰岛素分泌的影响

目的了解葡萄糖对INS-1细胞cAMP生成与胰岛素分泌的影响。方法用不同浓度的葡萄糖（2．8、7．5、15mmol／L）刺激INS-1细胞,作用30min后用ELISA方法检测cAMP与胰岛素的变化情况。结果葡萄糖浓度的提高对INS-1细胞cAMP生成与胰岛素分泌有明显的刺激作用（P〈0．05）,且表现出一定的浓度相关性。结论葡萄糖浓度提高能诱导INS-1细胞cAMP生成及促进其胰岛素分泌功能。     

Pre-steady state study of beta-adrenergic and purinergic receptor interaction in C6 cell membranes: undelayed balance between positive and negative coupling to adenylyl cyclase.

Interactions between beta-adrenergic and ADP purinergic receptors in C6 glioma cell membrane preparations were investigated under steady state and then pre-steady state conditions of adenylyl cyclase (EC 4.6.1.1) activity, in order to determine how fast the second receptor antagonizes the transduction mechanism of the first. Cell membranes were washed to deplete them as thoroughly as possible of low molecular weight compounds, especially ATP and ADP, and to ensure better control of both substrate and agonist nucleotide concentrations. ATP concentrations were kept constant with the use of an ATP-regenerating system; the C6 cell line exhibited very active ectonucleotidases. The purinergic agonist ADP was replaced by its nonhydrolyzable congener adenosine 5'-O-(2-thio)diphosphate (ADP beta S), which was demonstrated, like ADP, to inhibit isoproterenol-stimulated adenylyl cyclase activity in intact cells (IC50 for ADP, 0.5 +/- 0.1 microM; IC50 for ADP beta S, 25 +/- 2 microM) and in membrane preparations (IC50 for ADP beta S, 79 +/- 20 microM). In the case of membrane preparations, ADP beta S did not compete with ATP, the substrate of the cyclase-catalyzed reaction, and behaved apparently as a non-competitive inhibitor of the enzyme. The pre-steady state kinetics of isoproterenol-stimulated adenylyl cyclase activity measured with a pulsed quenched-flow apparatus have previously been shown to include two steps, the first very rapid (taking place within 1-2 sec) and giving rise to a burst of cAMP synthesis and the second much slower and corresponding to the steady state reaction. ADP beta S inhibited the occurrence of both steps with comparable IC50 values (mean value, 55 +/- 20 microM). In the presence of increasing concentrations of the purinergic receptor agonist, the time constant of the exponential burst reaction was not affected, but its amplitude progressively decreased to zero. These results showed that the extinction of the beta receptor cAMP response by the purinergic ADP receptor occurred within the dead-time of the pulsed quenched-flow apparatus, which was 50 msec. Such a rapid inhibition of cAMP production excluded modulation of isoproterenol-stimulated adenylyl cyclase activity by the ADP receptor by a pathway other than its direct negative coupling to the cyclase via a Gi protein. In this respect, the P2 purinergic ADP receptor of the C6 glioma cell line appears comparable to the P2t receptor of platelets.     

Effects of the beta-carbolines, harmane and pinoline, on insulin secretion from isolated human islets of Langerhans

It is well known that certain imidazoline compounds can stimulate insulin secretion and this has been attributed to the activation of imidazoline I(3) binding sites in the pancreatic beta-cell. Recently, it has been proposed that beta-carbolines may be endogenous ligands having activity at imidazoline sites and we have, therefore, studied the effects of beta-carbolines on insulin secretion. The beta-carbolines harmane, norharmane and pinoline increased insulin secretion two- to threefold from isolated human islets of Langerhans. The effects of harmane and pinoline were dose-dependent (EC(50): 5 and 25 microM, respectively) and these agents also blocked the inhibitory effects of the potassium channel agonist, diazoxide, on glucose-induced insulin release. Stimulation of insulin secretion by harmane was glucose-dependent but, unlike the imidazoline I(3) receptor agonist efaroxan, it increased the rate of insulin release beyond that elicited by 20 mM glucose (20 mM glucose alone: 253+/-34% vs. basal; 20 mM glucose plus 100 microM harmane: 327+/-15%; P<0.01). Stimulation of insulin secretion by harmane was attenuated by the imidazoline I(3) receptor antagonist KU14R (2 (2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole) and was reduced when islets were treated with efaroxan for 18 h, prior to the addition of harmane. The results reveal that beta-carbolines can potentiate the rate of insulin secretion from human islets and suggest that these agents may be useful prototypes for the development of novel insulin secretagogues.     

Effects of alpha-adrenoceptor antagonists on clonidine-induced inhibition of insulin secretion by isolated pancreatic islets.

The effects of clonidine, yohimbine, corynanthine and prazosin on glucose-induced insulin secretion by incubated or perifused mouse pancreatic islets were investigated. Clonidine (0.1 microM) inhibited glucose-induced insulin secretion alone and in the presence of yohimbine (0.1 microM), corynanthine (10 microM) or prazosin (1 microM). In higher concentrations, yohimbine (1-10 microM) antagonized the inhibitory effect of clonidine (0.1 microM) upon glucose-induced insulin secretion by incubated islets and by perifused islets. The results support the view that adrenergic inhibition of insulin secretion is mediated by alpha 2-adrenoceptors on pancreatic beta-cells.     

Phentolamine reverses NPY-induced inhibition of insulin secretion in isolated rat islets

It has been shown that the rodent pancreas is innervated by neuropeptide Y (NPY) nerves, some of which are adrenergic, and that NPY inhibits glucose-induced insulin secretion in vivo in the mouse and that from isolated rat islets in vitro. We now investigated whether the alpha-adrenoceptor antagonist phentolamine affects the inhibitory action of NPY on insulin secretion from isolated rat islets. It was found that NPY dose dependently inhibited insulin secretion stimulated by glucose (11.1 mM). At a concentration of 10(-7) M, NPY totally abolished the insulin secretory response to glucose. It was also found that incubation with the alpha-adrenoceptor antagonist phentolamine (10(-6) M) itself enhanced the insulin secretion at 3.3 mM but not at 16.7 mM glucose. Moreover, phentolamine counteracted the inhibitory action of NPY. Thus, at 10(-8) M, NPY could no longer inhibit insulin secretion when phentolamine (10(-6) M) was present, whether 3.3 mM or 11.1 mM glucose was present. In contrast, somatostatin (10(-7) M) could inhibit insulin secretion, both in the presence and absence of phentolamine (10(-6) M); this showed that phentolamine does not reverse all types of inhibition. However, when the dose of phentolamine was decreased to 10(-7) M, the inhibitory action of NPY on glucose-induced insulin secretion was retained indicating that the reversal of the NPY effect by phentolamine is a competitive effect. It is concluded that NPY inhibits glucose-induced insulin secretion by a direct action on the islets, and that phentolamine reverses this inhibitory action of NPY in a competitive manner.