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.     

Adenine nucleotide pattern in rat pancreatic islets exposed to nutrient secretagogues

The effects of d-glucose, d-mannose, d-galactose, d-glyceraldehyde, pyruvate, l-lactate, 2-ketoisocaproate, l-leucine, and/or l-glutamine on the ATP and ADP content of rat isolated pancreatic islets were reevaluated in order to compare changes evoked by these nutrient secretagogues in the islet ATP content and ATP/ADP ratio to their effects upon insulin release. Although being compatible with the fuel concept for nutrient-stimulated insulin secretion, the results of this study also argue against the monolithic view that the adenine nucleotide pattern in islet cells represents the sole coupling factor between metabolic and more distal events in the process of nutrient-stimulated insulin release.     

Neurotensin inhibits glucose but not glucagon-induced insulin and somatostatin release in isolated islets.

The effects of neurotensin on insulin and somatostatin release were examined in isolated pancreatic islets prepared from 3-4 days rats, and maintained in culture for 48 h before use. In the presence of 12 mM glucose, glucagon (50-2,000 ng/ml, i.e. 14-560 nM) caused a 2-fold increase in insulin and somatostatin release. Neurotensin (150 ng/ml, i.e., 100 nM) did not affect the glucagon-stimulated release, nor did it alter the release of either peptide measured at 12 mM glucose in the absence of glucagon. In contrast, neurotension markedly inhibited the release of both insulin and somatostatin that was induced by 23 mM glucose. These observations suggest that neurotensin may modulate the release of insulin and somatostatin evoked by high glucose concentrations, but not that resulting from the action of glucagon on pancreatic islets.     

Effect of low concentrations of glucagon on insulin release and cyclic AMP content in isolated rat islets

Studies on hormone action in isolated islets have generally been carried out using concentrations far above physiologic levels. This study investigates whether glucagon at concentrations close to the physiologic level is insulinotropic in isolated islets and how this relates to islet cyclic AMP levels. Collagenase isolated rat islets were tested directly after isolation or after a 24-hour tissue culture. Insulin release and islet cyclic AMP content were determined during a 30-minute incubation by radioimmunoassay. After maintenance in tissue culture glucose-induced (16.7 mmol/L) insulin release was clearly enhanced by glucagon concentrations between 2 and 1,000 ng/mL in a dose-related manner. Islet cyclic AMP was increased only by glucagon 1 mumol/L (3.8 micrograms/mL). When phosphodiesterases were inhibited (0.1 mmol/L 3-isobutyl-1-methylxanthine) insulin release was stimulated by 1 ng/mL and cyclic AMP by 100 ng/mL. By contrast, in freshly isolated islets, glucagon concentrations in the range of 1 to 100 micrograms/mL were needed to augment glucose-induced insulin release. These findings demonstrate that the hormone sensitivity of collagenase isolated islets is markedly improved by short-term maintenance in tissue culture. The threshold level for a detectable effect on islet cyclic AMP is considerably higher than for glucose-induced insulin release. Since in hepatocytes two signal transduction systems for glucagon have recently been demonstrated, the results could mean that at low concentrations glucagon effects may not be mediated via cyclic AMP.     

Evidence that somatostatin inhibits proinsulin synthesis and insulin release by isolated pancreatic islets of the rat

Conflicting reports on the direction and magnitude of the effect of somatostatin on (pro)insulin synthesis prompted our investigation. Two assays for proinsulin synthesis were designed in which [4,5-3H]-L-leucine incorporation into proinsulin was normalized on the basis of postincubation insulin levels rather than on the number of islets incubated. Somatostatin at a concentration of 10 micrograms/ml inhibited 300 mg/dl glucose-stimulated proinsulin synthesis by 25% from 448 +/- 25 dpm/microunits insulin to 336 +/- 25 dpm/microunits insulin (disintegrations per minute in the proinsulin peak per microunit extractable insulin) (p less than 0.05). Glucagon (10 micrograms/ml) reversed the inhibitory effect of somatostatin on proinsulin synthesis from 336 +/- 25 dpm/microunits insulin to 480 +/- 44 dpm/microunits insulin (p less than 0.02). Somatostatin (10 micrograms/ml) had no significant effect on proinsulin synthesis in the presence of 70 mg/dl or 150 mg/dl glucose. Insulin release in 300 mg/dl glucose was inhibited 38% by 10 micrograms/ml somatostatin from 3.05 +/- 0.40 mU medium/mU tissue to 1.90 +/- 0.10 mU medium/mU tissue (p less than 0.01) over a 45-minute incubation period. These data suggest that somatostatin may act on glucose signal transduction on a level at which both insulin synthesis and secretion are affected. Further, the results are consistent with the hypothesis that cyclic AMP participates in mediating somatostatin effects on B-cell metabolism.     

The effects of vitamin A on insulin release and glucose oxidation in isolated rat islets.

We tested the effects of vitamin A, a membrane surface-active agent, on glucose (16.7 mM)-induced biphasic insulin release from collagenase-isolated rat islets. Also, efforts were made to correlate the effects of vitamin A with glucose oxidation. Vitamin A (10(-4) M) inhibited first- and second phase insulin release; 10(-5) M vitamin A inhibited second phase release only and to a lesser extent than that observed with 10(-4) M vitamin A; and 10(-6) M vitamin A had no effect. Vitamin A (10(-7) M) stimulated biphasic insulin release. Exposure to high glucose (27.8 mM) overcame the effects of 10(-4) M vitamin A on first phase release, but not on second phase release of insulin. Exposure to 10(-5) M hydrocortisone opposed the effects of 10(-4) M vitamin A on both phases of insulin release. Vitamin A (10(-4) and 10(-5) M) inhibited glucose oxidation by islets, as measured by the production of 14CO2 from [14C]glucose. The effects of vitamin A on insulin release were dissociated in part from those effects on glucose oxidation, in that hydrocortisone opposed the effect of vitamin A on insulin release but not on glucose oxidation. The effects of vitamin A on insulin secretion can best be explained by the interaction of vitamin A at multiple sites affecting the membrane and intracellular glucose oxidation.     

Stimulation of insulin release in isolated rat islets by GIP in physiological concentrations and its relation to islet cyclic AMP content

Summary Several insulinotropic hormones have been shown to increase the level of cyclic AMP in isolated islets. This study was performed to investigate whether gastric inhibitory polypeptide (glucose-dependent insulin-releasing polypeptide) has a similar effect, in particular at concentrations close to the physiological level in blood. Collagenase isolated rat islets were maintained for 24 h in tissue culture (medium 199) and then incubated for 30 min for measurement of insulin release and cyclic AMP content. Glucose-induced (16.7 mmol/ 1) insulin release was enhanced by gastric inhibitory polypeptide 1–100 ng/ml (0.196–19.6 nmol/l) in a dose-related fashion. The cyclic AMP content was enhanced only by 100 ng/ ml. However, when 0.1 mmol/l of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine was present, even 1 ng/ ml of gastric inhibitory polypeptide increased both cyclic AMP content and insulin release. Such a concentration of the hormone can be measured in human blood after a meal. In contrast, in freshly isolated islets no effect of the hormone on glucose-induced insulin release or cyclic AMP content could be detected for concentrations ranging from 1 to 100 ng/ml. These findings demonstrate that the hormone sensitivity of isolated islets is markedly enhanced by short-term maintenance in tissue culture. The results suggest that an increase in cyclic AMP is seen in response to gastric inhibitory polypeptide and may be causally related to the insulinotropic effect of the hormone.     

Beta-adrenergic receptor agonists and cyclic nucleotide phosphodiesterase inhibitors: shifting the focus from inotropy to cyclic adenosine monophosphate.

Clinical trials of beta-adrenergic receptor agonists and cyclic nucleotide phosphodiesterase inhibitors in heart failure have demonstrated a reduction in survival in treated patients despite initial inotropic responses. These findings have led many to infer that activation of the mechanisms through which contractility is increased has deleterious effects on failing myocardium. It should be remembered, however, that these agents act proximately by raising intracellular cyclic adenosine monophosphate (cAMP) content and stimulating protein phosphorylation by cAMP-dependent protein kinase, and that the proteins whose phosphorylation contributes to the inotropic responses may be different from the proteins whose phosphorylation contributes to the reduction in survival. Evidence in support of the latter interpretation is presented, and potential therapeutic approaches through which the phosphorylation of different proteins might be selectively affected are considered.     

Muscarinic cholinergic induced secretin subsensitivity in rat isolated pancreatic acini. Effects on amylase release, cyclic adenosine monophosphate and inositol phosphate formation

In this study, dispersed rat pancreatic acini exhibited secretin subsensitivity in their capacity to release amylase after preexposure to increasing concentrations of the muscarinic cholinergic agonist carbamylcholine. The present study also explores the potential mechanisms involved in this cellular desensitization phenomenon. Secretin subsensitivity of pancreatic acini pre-exposed to 10(-4) M carbamylcholine was observed only at secretin concentrations above 10(-8) M. The desensitized cells had not recovered 3 h after the cholinergic agonist exposure. In these acini, the adenylate cyclase pathway remained unaltered because cholera toxin, forskolin, and 8-Br-cAMP still induced weak, but normal, amylase release when compared with control acini. In vivo administration of pertussis toxin failed to protect the dispersed pancreatic acini against carbamylcholine-induced secretin subsensitivity. Moreover, cAMP production by these acini in response to secretin, cholera toxin, and forskolin was similar to that observed in control acini. Secretin stimulation of inositol phosphate (InsP1, InsP2, InsP3) production after carbamylcholine pre-exposure remained equivalent to that observed in acini that had never been exposed to the cholinergic agonist. Thus, after muscarinic cholinergic agonist exposure, pancreatic acini showed secretin subsensitivity in their capacity to release enzyme. This phenomenon appears to result from modifications at post-second messenger loci.     

alpha-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.

We determined the effect of alpha-adrenergic receptor stimulation on cyclic adenosine monophosphate (cyclic AMP) concentrations in isolated myocytes derived from adult rat hearts and in isolated perfused rat hearts. Activation of alpha-adrenergic receptors with either phenylephrine (10(-8) M to 10(-6) M) or epinephrine (10(-8) M to 10(-6) M) plus propranolol (10(-6) M) resulted in a reduction in cyclic AMP levels in isolated myocytes. The action of phenylephrine was antagonized by phentolamine (10(-6) M). Phenylephrine (10(-5)M attenuated cyclic AMP generation in response to isoproterenol (10(-8) M and 10(-5) M). However, this effect of phenylephrine was not antagonized by phentolamine. Elevation of cyclic AMP concentrations produced by glucagon and by theophylline in isolated myocytes was attenuated by phenylephrine and by epinephrine plus propranolol and the attenuation was antagonized by phentolamine. In isolated perfused rat hearts epinephrine (10(-6) M), when given with propranolol, diminished the rate of development of tension and also reduced tissue levels of cyclic AMP. Epinephrine alone, as well as isoproterenol, increased contractility and myocardial cyclic AMP concentrations as expected. These results indicate that catecholamines may increase or decrease cyclic AMP levels in rat myocardium, depending on the intensity of stimulation of receptor types. Increases are mediated by beta-adrenergic receptors, whereas decreases appear to by mediated by alpha-adrenergic receptors.     

Effects of forskolin on insulin release and cyclic AMP content in rat pancreatic islets

The effects of forskolin, a diterpene stimulator of adenylate cyclase, on cyclic AMP content and insulin release, have been studied in rat pancreatic islets. In the presence of 2.8 mM glucose, 0.3 to 30 microM forskolin gave concentration-dependent increases in cyclic AMP content to as much as seven-fold without causing any significant increase in insulin release. With 5.6 mM glucose, cyclic AMP contents were elevated by forskolin to the same extent as with 2.8 mM glucose but, under these conditions, insulin release was stimulated. 3 microM forskolin was a maximal concentration with respect to insulin release, but not for the elevation of cyclic AMP levels. Concentration-dependent increases in both insulin release and cyclic AMP content were detected over the range of 0.3 to 3 microM forskolin. The effect of forskolin to stimulate insulin release was rapid and was reversible within 15 minutes of removal of the drug. When tested with 16.7 mM glucose, forskolin potentiated both phases of stimulated release, with the greater effect upon the first phase.     

Fentanyl inhibits glucose-stimulated insulin release from β-cells in rat pancreatic islets

AIM： TO explore the effects of fentanyl on insulin release from freshly isolated rat pancreatic islets in static culture. METHODS： Islets were isolated from the pancreas of mature Sprague Dawley rats by common bile duct intraductal collagenase V digestion and were purified by discontinuous Ficoll density gradient centrifugation. The islets were divided into four groups according to the fentanyl concentration： control group （0 ng/mL）, group I （0.3 ng/mL）, group I （3.0 ng/mL）, and group III （30 ng/mL）. In each group, the islets were co-cultured for 48 h with drugs under static conditions with fentanyl alone, fentanyl ＋ 0.1 μg/mL naloxone or fentanyl ＋ 1.0 μg/mL naloxone. Cell viability was assessed by the MTT assay. Insulin release in response to low and high concentrations （2.8 mmol/L and 16.7 mmol/L, respectively） of glucose was investigated and electron microscopy morphological assessment was performed. RESULTS： Low- and high-glucose-stimulated insulin release in the control group was significantly higher than in groups I and II （62.33 ± 9.67 μIU vs 47.75 ± 8.47 μIU, 39.67 ± 6.18 μIU and 125.5 ± 22.04 μIU vs 96.17 ± 14.17 μIU, 75.17 ± 13.57 μIU, respectively, P 〈 0.01） and was lowest in group III （P 〈 0.01）. After adding 1 μg/mL naloxone, insulin release in groups II and II was not different from the control group. Electron microscopy studies showed that the islets were damaged by 30 ng/ml fentanyl. CONCLUSION： Fentanyl inhibited glucose-stimulated insulin release from rat islets, which could be prevented by naloxone. Higher concentrations of fentanyl significantly damaged β-cells of rat islets.     

Alloxan-induced alteration of insulin release,rubidium efflux and glucose metabolism in rat islets stimulated by various secretagogues

Summary Insulin release and⁸⁶Rb efflux were studied in perifused rat islets exposedin vitro to alloxan (2 mmol/l) for 5 min. At a low glucose concentration, alloxan transiently increased⁸⁶Rb efflux. Alloxan immediately and completely abolished the secretory response to glucose (15 mmol/l) and markedly delayed the reduction in⁸⁶Rb efflux normally produced by the sugar. 3-O-methylglucose (20 mmol/l) provided complete protection against the alteration of⁸⁶Rb efflux and partial protection against the inhibition of insulin release. Immediately after alloxan treatment, glyceraldehyde,-ketoisocaproic acid and tolbutamide still induced a rapid release of insulin, but the late phase normally stimulated by glyceraldehyde and-ketoisocaproic acid was inhibited. If islets were exposed to glyceraldehyde or tolbutamide 15 min after alloxan treatment, the rapid insulin release was also markedly impaired. Alloxan failed, however, to affect the ability of these three stimuli to reduce⁸⁶Rb efflux from islet cells. Glucose oxidation and utilization were decreased in alloxan-treated islets and 3-O-methylglucose protected against this effect. The results show that the glucose recognition system in B-cells is the most rapidly and severely affected by alloxan. The drug also alters the response to other secretagogues, the insulin releasing properties of which can be impaired without alteration of their ability to reduce⁸⁶Rb efflux.     

3,5 cyclic adenosine monophosphate mediates the salmon calcitonin-induced increase in hypothalamic tyrosine hydroxylase activity.

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1-10 nM) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nM) increased TH activity to 150-175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mM and 5 microM, respectively. sCT (10 nM) increased radiolabeled phosphate incorporation into TH protein to 169% of control values and 1 mM Rp-cAMPS completely blocked this effect. In contrast, neither Calphostin C, a protein kinase C inhibitor, nor U-73122, a phospholipase C inhibitor, significantly altered the ability of sCT to increase TH activity. Likewise, the sCT-induced increase in TH activity was observed after pretreating the cells with either BAPTA/AM, an intracellular calcium chelator, or thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump. These data indicate that sCT has a profound stimulatory effect on TH activity in fetal hypothalamic cells and that enhanced phosphorylation of TH coincides with the sCT-induced increase in enzyme activity. Moreover, CT receptors, which are linked to cAMP production, are expressed in the hypothalamic cells and a cAMP-dependent mechanism mediates the sCT-induced activation and phosphorylation of TH.     

Ghrelin在离体大鼠胰岛中抑制胰岛素分泌和上调Kir6．2表达

目的明确ghrelin在离体大鼠胰岛中对胰岛素分泌和内向整流钾通道（Kit6．2）表达的影响,并讨论两者的关系。方法离体大鼠胰岛以高浓度葡萄糖及不同浓度ghrelin和（或）其受体拮抗剂ED-lys^3]-GHRP-6孵育1h,采用放免法测定上清液的胰岛素,采用RT-PCR检测Kir6．2、磺酰脲受体1（SUR-1）、解偶联蛋白2（UCP-2）、葡萄糖转运子2（GluT-2）、胰十二指肠同源盒1（PDX-1）等基因的表达。结果10。～10“mol／Lghre｜in呈剂量依赖性抑制离体大鼠胰岛高浓度葡萄糖刺激的胰岛素释放,且剂量依赖性增加Kir6．2mRNA表达,但对SUR-1、UCP-2、GluT-2及PDX-1 mRNA表达则无显著影响。ED-lys^3]-GHRP-6可消除ghrelin对Kir6．2 mRNA表达的上调作用。结论在离体大鼠胰岛中,ghrelin通过作用于其受体,促进ATP敏感性钾通道组成成分Kir6．2的表达,改变钾通道功能状态。这可能是ghrelin抑制葡萄糖刺激的胰岛素分泌的机制之一。     

Interaction of cyclic AMP and alloxan on insulin secretion in isolated rat islets perifused in vitro.

The interaction of alloxan and cyclic AMP on glucose-induced insulin secretion was studied with the use of isolated rat islet perifusion. Simultaneous perifusion with cyclic AMP and alloxan did not protect the islets against the effect of alloxan. However, addition of dibutyryl cyclic AMP to the alloxan solution produced 40% protection of glucose-induced insulin secretion. Partial protection was obtained with either theophylline (41%) or caffeine alone (54%), and the addition of 1 mg/ml glucose to the theophylline or caffeine solution provided greater than 68% protection. The levels of islet tissue cyclic AMP were more than 2.1 times that of islets not protected against the alloxan effect, when partial or nearly complete reversal of the inhibitory action of alloxan on glucose-induced insulin secretion was effected by theophylline or caffeine. These results suggest that cyclic AMP affords partial protection against the effect of alloxan on glucose-induced insulin secretion.     

Endothelin-1 and endothelin-3 stimulate insulin release by isolated rat pancreatic islets

Endothelins (ETs) are potent vasoconstrictive peptides released from the endothelium and other tissues, which act on target cells by receptorial calcium-mediated mechanisms. ET-1 levels are increased in diabetes, and observations suggest the involvement of ETs in the pathogenesis of diabetic angiopathy. However, it is not possible to exclude that ETs might also influence insulin secretion or function. In vivo infusion of ET-1 in rats induces hypoglycaemia and hyperinsulinemia and in vitro incubation with ET-1 stimulates insulin release by mouse islets. Therefore, ETs might be involved in a circulus vitiosus, resulting in hyperinsulinemia and diabetic angiopathy. The purpose of our study was to verify the effect of ET-1 on rat islets, in both the presence and absence of physiological glucose concentration. Moreover, we tested the effect of another isoform of endothelins, ET-3, and verified the involvement of extracellular calcium in such events. Islets were incubated with increasing ET-1 or ET-3, with or without glucose 5.6 mM. Other samples were prepared using calcium-free medium. Incubation in medium containing ET-1 and ET-3, in the presence of glucose and calcium, induced an increase in insulin release. When ET-1 and ET-3 were incubated without glucose and calcium, insulin release was not modified. Our studies demonstrate that: 1) ET-3, like ET-1, stimulates insulin release by rat isolated islets; 2) direct insulin stimulating effect on islets of both ET-1 and ET-3 is evident with physiological glucose concentrations and is calcium mediated. These results support the hypothesis of ET involvement in the regulation of insulin secretion.     

Possible involvement of cholinergic nicotinic receptor in insulin release from isolated rat islets

Insulin release is influenced by the autonomic nervous system. Regarding parasympathetic control, previous reports have shown that regulation of insulin release is executed exclusively through muscarinic receptors in the pancreatic islets. In the present study, however, we examined the effect on insulin release at the islet level of various agents affecting the parasympathetic nervous system, especially nicotinic receptor blockers. Pancreatic islets isolated from adult Wistar male rats were incubated with these agents and insulin release in the media was measured. Acetylcholine chloride (10(-5) M), as well as distigmine bromide (10(-6), 10(-5) M), both of which are cholinesterase inhibitors, stimulated insulin release, whereas atropine (5 x 10(-6), 5 x 10(-5) M) suppressed it. On the other hand, serum and IgG from myasthenia gravis patients, containing anti-acetylcholine receptor antibodies, affected insulin release, and alpha-bungarotoxin (10(-9)-10(-7) M), a nicotinic receptor blocker, stimulated insulin release dose-dependently. The present observations suggest that insulin release is influenced by the parasympathetic nervous system, mediated via not only muscarinic but also nicotinic receptors.     

Interaction between cyclic adenosine monophosphate and cyclic gunaosine monophosphate in guinea pig ventricular myocardium.

The purpose of this study was to examine the mechanisms underlying adrenergic-cholinergic antagonism in ventricular myocardium. Myocardial contractility, cyclic adenosine monophosphate (AMP) levels, and cyclic guanosine monophosphate (GMP) levels were measure in isolated guinea pig ventricles after treatment with various inotropic agents given alone and simultaneously with acetylcholine. Acetylcholine alone markedly elevated cyclic GMP levels but did not substantially change myocardial contractility. However, the same concentration of acetylcholine significantly attenuated the inotropic effect of isoproterenol and histamine, two drugs that may act by increasing myocardial levels of cyclic AMP. The decrease in the inotropic response to isoproterenol did not appear to be due to a decrease in the generation of cyclic AMP, because cyclic AMP levels were similar in hearts receiving isoproterenol alone and those receiving isoproterenol with acetylcholine. Dibutyryl cyclic GMP also antagonized the intropic action of isoproterenol. Acetylcholine did not alter the inotropic effects of ouabain, an agent that increases myocardial contractility without changing cyclic AMP levels. These results suggest that cyclic GMP mediates the antiadrenergic effects of acetylcholine by specifically antagonizing the inotropic actions of cyclic AMP.