Effects of amidated rat islet amyloid polypeptide on glucose-stimulated insulin secretion in vivo and in vitro in rats

Amidated rat islet amyloid polypeptide (IAPP) is a constituent of secretory granules in islet B-cells. We examined its influence on glucose-stimulated insulin secretion in rats. In vivo, intravenous infusion of IAPP (68 pmol/min) did not affect glucose-stimulated insulin secretion. In vitro, IAPP slightly inhibited glucose (8.3 mM)-stimulated insulin secretion from isolated rat islets at the high dose level of 10(-5) M but not at the lower dose levels. We conclude that the IAPP is not involved in the normal regulation of insulin secretion.     

Similar effects of succinic acid dimethyl ester and glucose on islet calcium oscillations and insulin release

The relative contribution of glycolysis vs. oxidative metabolism to the stimulus secretion coupling mechanism of beta-cells was investigated in isolated islets. For that purpose, the secretory and intracellular calcium responses of islets to both glucose and succinic acid dimethyl ester (SAD) were compared. After 45 min of rat islet perifusion in the absence of substrates, the maximum secretory responses to glucose (20 mmol/L) and SAD (10 mmol/L) were qualitatively and quantitatively indistinguishable. Malonic acid dimethyl ester (a permeable citric acid cycle inhibitor) suppressed the insulin secretory response to both 20 mmol/L glucose and 10 mmol/L SAD (-70% on average). The inhibitor decreased within 70% the rate of 14CO2-production from 10 mmol/L [2-(14)C]pyruvate without affecting the rate of 20 mmol/L D-[5-(3)H]glucose utilization. Both, 11.1 mmol/L glucose and 10 mmol/L SAD, elevated the intracellular calcium concentration and induced a similar pattern of oscillations that were rapidly ablated by 20 mmol/L malonic acid dimethyl ester. However, the intracellular concentration of calcium declined to basal values several minutes after the introduction of the inhibitor in the presence of SAD whereas it remained elevated in the case of glucose. In conclusion: (1) An exclusive increase of mitochondrial metabolism in pancreatic islets was sufficient to mimic the effects of glucose on intracellular calcium and insulin secretion. (2) Islet glycolysis and/or the re-oxidation of cytoplasmic NADH allowed the maintenance of an elevated, though non-oscillating, intracellular calcium concentration, but a reduced response to glucose.     

In vivo and in vitro evaluation of a solid dispersion system of gliclazide:PEG 6000

OBJECTIVE: Gliclazide is a potent antidiabetic agent because of its capability to decrease blood glucose level via stimulating endogenous insulin secretion from beta-pancreas cells. Gliclazide is insoluble in water and has low dissolution rate. In this study, polyethylene glycol (PEG) 6000 was used as a matrix to disperse gliclazide in the solid state, and the pharmacokinetic profile of this solid dispersion was studied in rats. DESIGN: The solid dispersion of Gliclazide:PEG 6000 (1:4) was prepared by solvent evaporation method. MAIN OUTCOME MEASURES: Samples characterization included differential scanning calorimetry (DSC), infrared spectroscopy (IR), X-ray diffraction (XRD), and solubility and dissolution test. In vivo study was carried out in healthy rats, randomly. After a single dose of oral administration, blood samples were collected pre-dose (15 min before) and 1, 2, 3, 4, 5, 6, 8, 10, and 12 h post-dose. Plasma concentration of gliclazide was determined by high pressure liquid chromatography method using C-18 column, with mobile phase KH2PO4 (pH 4.6)-acetonitril (40:60 v/v) and UV detection at 229 nm. RESULTS: Results showed that there were no differences in DSC, IR spectroscopy, XRD, and dissolution test between the solid dispersion and physical mixture. In vivo data showed that the Tmax of gliclazide in solid dispersion and physical mixture was significantly decreased, while the Cmax, AUC(0-12), and AUC(0-infinity) were significantly increased compared to gliclazide alone. These results indicate that the rapid Tmax was due to rapid absorption of gliclazid across the GI tract membrane. Increased Cmax, AUC(0-12), and AUC(0-infinity) indicate a better absorption of gliclazide in solid dispersion and physical mixture than of gliclazide alone. CONCLUSION: Increased in gliclazide dissolution in the presence of PEG 6000 was followed by improved in vivo data.     

门冬胰岛素30强化治疗对初诊2型糖尿病患者胰岛功能的影响

目的:探讨短期门冬胰岛素30强化治疗对初诊2型糖尿病患者胰岛β细胞功能的影响。方法:对30例初诊2型糖尿病患者每日注射2次门冬胰岛素30,强化治疗2周,治疗前后分别进行口服葡萄糖耐量试验(OGTT)及胰岛素释放试验,测定0,30,120 min的血糖和胰岛素值,分别以稳态模型法β细胞功能指数(HOMA-β)评估β细胞功能,以稳态模型法胰岛素抵抗指数(HOMA-IR)评估胰岛素抵抗状况,以早相胰岛β细胞分泌指数(△I30/△G30,糖负荷30min净增胰岛素与净增葡萄糖的比值)评估早期胰岛素分泌能力。结果:经过短期门冬胰岛素30强化治疗后,患者空腹血糖(FPG)、餐后2 h血糖(2 hPG)、HOMA-IR均明显下降(P<0.05);而HOMA-β、△I30/△G30显著升高(P<0.001)。结论:短期门冬胰岛素30强化治疗能够明显改善初诊2型糖尿病患者胰岛分泌功能,减轻胰岛素抵抗,改善糖代谢。     

Effect of different stimulators and a blocker of insulin release on islet Na+, K+-ATPase activity

The effect of several insulin secretagogues and a blocker upon islet Na+, K+-ATPase activity was studied using rat islet homogenates. None of the agents tested modified the enzyme activity when added directly to the enzyme assay. Activity of Na+, K+-ATPase measured in islets preincubated during 3 min with glucose 3.3, 8 or 16.6 mM, as well as with 15 mM KIC or 1.2 microM somatostatin, did not significantly change. The presence of glucagon (1.4 microM) plus theophylline (10 mM) in the preincubation medium significantly enhanced activity while tolbutamide (1.48 mM) or gliclazide (76 microM) significantly decreased such activity. These results suggest that Na+, K+-ATPase activity would not be a main common step involved in the mechanism by which glucose, KIC, glucagon + theophylline and somatostatin exert their effect on insulin secretion. Conversely, the enzyme might contribute to the stimulatory effect of gliclazide and tolbutamide on insulin release. Such effect would be secondary to the release of some cellular mediator rather than a direct action of these compounds on the enzyme. Such effect would later favor a rise in the cytosolic concentration of calcium which might trigger the release of insulin.     

Effect of middle-term gliclazide treatment on insulin secretion in non-insulin dependent diabetics

Summary

Insulin secretion was studied in 12 non-insulin dependent diabetics during middle-term administration of the sulphonylurea gliclazide. Blood sugar, C-peptide and glucagon were also estimated during the intravenous glucose tolerance and arginine tests performed before and after therapy. After 3 months of gliclazide/therapy (240?mg/day) in addition to a low carbohydrate diet, the intravenous glucose tolerance test showed a significant reduction in blood sugar levels and in the partial and total areas under the blood sugar curve, as well as an improvement in early insulin secretion, characterized by a significant increase in plasma C-peptide at 4, 10 and 20 minutes. Plasma glucagon levels were not affected by the sulphonylurea therapy. In the arginine test, blood sugar levels were lower at the end of the treatment period; plasma insulin, C-peptide and glucagon did not change significantly. In this study, plasma C-peptide has proved to be a better indicator of stimulated insulin secretion than plasma insulin levels. The scarcity of hypo-glycaemic episodes during therapy with gliclazide may be related to the selective stimulation of early insulin secretion by this drug.     

The effects of ingestion time of gliclazide in relationship to meals on plasma glucose,insulin and c-peptide levels

Summary The effect of altering the timing of gliclazide administration in relation to a meal was studied in ten type 2 (non-insulin dependent) chronically treated diabetics. Gliclazide was given 30 min before, at the start of and 30 min after breakfast or omitted altogether. Plasma gliclazide was present at greater than 2 mg/l throughout the study periods. Administration at 30 min after the meal significantly delayed the time to peak for plasma gliclazide. No significant difference was noted in plasma glucose, insulin or c-peptide patterns with any protocol. It is concluded that, in clinical practice, with chronically treated diabetics the timing of gliclazide ingestion in relation to meals is not critical.     

促泌剂对胰岛素强化治疗后的初诊2型糖尿病患者胰岛β细胞分泌胰岛素的影响

目的:观察瑞格列奈或格列美脲对短期胰岛素强化治疗后血糖达标的初诊2型糖尿病(T2DM)患者胰岛β细胞分泌胰岛素的影响。方法:对60例经为期约2周的胰岛素强化治疗后血糖达标的初诊T2DM患者,进行75 g葡萄糖耐量试验(OGTT),测定血糖和胰岛素值,计算早相胰岛β细胞分泌指数(△I30/△G30,糖负荷30 min净增胰岛素与净增葡萄糖的比值),以30～120 min的胰岛素分泌曲线下面积和葡萄糖曲线下面积比值(△AUCI 30-120/△AUCG 30-120)表示晚相胰岛素分泌能力。然后按照1∶1比例,随机分成瑞格列奈组和格列美脲组,分别接受瑞格列奈和格列美脲治疗,维持6个月后重复行OGTT,检测上述指标。对口服药物治疗前后胰岛β细胞功能进行自身比较。结果:瑞格列奈组△I30/△G30差异有显著性(P<0.01),而△AUCI 30-120/△AUCG 30-120无统计学意义(P>0.05)。格列美脲组△AUCI 30-120/△AUCG 30-120显著上升(P<0.05),而△I30/△G30无统计学意义(P>0.05)。结论:瑞格列奈以促进早相分泌为主,格列美脲以促进晚相分泌为主。     

Histamine release from saponin-permeabilized rat mast cells by calcium

The first effect of receptor activation on the mast cell surface, initiating histamine secretion, is an increase in the cytosol Ca2+ concentration. It should then be possible to induce histamine secretion by calcium alone, if the calcium permeability of the cell membrane could be increased without any significant interference with the physiological cell functions. This was achieved in the present study by adding low concentrations of saponin (0.0005% and 0.001% w/v) to the medium. When calcium was added to the saponin-permeabilized cells, around 40% histamine release occurred with 0.25 mM extracellular calcium (free Ca2+ 0.15 mM). The release was inhibited by antimycin A (1 microM). Transmission electron microscopy showed formation of vacuoles containing granules stripped of their membranes, which characterize a secretory response. The observations are consistent with a limited increase in the calcium permeability of the cell membrane for a brief period. There was apparently an increase in the cytoplasmic calcium concentration, which acted through calmodulin, since the histamine release induced by calcium from the permeabilized mast cells could be inhibited by a calmodulin-antagonist, mepacrine (10-30 microM).     

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。     

The inhibitory effect of cadmium on the secretory activity of the isolated perfused rat pancreas

Perfusion of the isolated rat pancreas with cadmium (Cd) (1 × 10^?3 and 5 × 10^?4m) inhibits the insulin secretory response to glucose (300 mg/100 ml), tolbutamide (40 mg/100 ml), and potassium ions (30 mEq/liter). Cadmium inhibition of pancreatic secretory activity is immediate in onset, and not reversed by simple washout of the organ with perfusion medium. Perfusion of the inhibited organ with a combination of glucose and theophylline results in partial reversal of the inhibition. It is suspected that Cd-induced inhibition of insulin secretion may be mediated through interference with calcium uptake by the pancreatic beta cell.     

Serotonin-induced secretion of von Willebrand factor from human umbilical vein endothelial cells via the cyclic AMP-signaling systems independent of increased cytoplasmic calcium concentration

Endothelial cells are able to synthesize von Willebrand factor (vWf) protein, which is then either secreted in a constitutive way or stored within specific cellular secretory granules, the Weibel-Palade bodies. Stimulated secretion of vWf from these organelles is thought to be induced by agonists causing a transient increase in cytoplasmic calcium concentrations. Serotonin (5-hydroxytryptamine, 5-HT), a local transmitter substance released by activated platelets, has also recently been shown to induce the secretion of vWf. In experiments with human umbilical vein endothelial cells (HUVEC), we found that the 5-HT-induced secretion occurred without a significant increase in cellular calcium levels. The 5-HT 1(D) subtype-specific receptor agonist sumatriptan also induced the release of vWf without causing a calcium signal in HUVEC. Stimulation of endothelial cells with the adenylate cyclase inhibitor, MDL-12 A330, led to the secretion of vWf as well. Simultaneous addition of submaximal concentrations of histamine and 5-HT to HUVEC potentiated the effects of either agonist. Together, these results suggest that in HUVEC 5-HT-induced secretion of vWf is mediated by a decrease in cyclic AMP levels and is independent of changes in cytoplasmic calcium levels.     

Alterations of insulin response to different beta cell secretagogues and pancreatic vascular resistance induced by N omega-nitro-L-arginine methyl ester.

1. We studied a possible interplay of pancreatic NO synthase activity on insulin secretion induced by different beta cell secretagogues and also on pancreatic vascular bed resistance. 2. This study was performed in the isolated perfused pancreas of the rat. Blockage of NO synthase was achieved with Nw-nitro-L-arginine methyl ester (L-NAME); The specificity of the antagonist was checked by using its D-enantiomer as well as by substitutive treatments with sodium nitroprusside (SNP) as a NO donor in studies of glucose-induced insulin secretion. 3. Arginine (5 mM) induced a monophasic response which was, in the presence of L-NAME at equimolar concentration, very strongly potentiated and converted into a 13 times higher biphasic one. D-NAME (5 mM) was only able to induce a 3 times higher response, but provoked a similar vasoconstrictor effect. 4. The small biphasic insulin secretion induced by L-leucine (5 mM) was also strongly enhanced, by 8 times, in the presence of L-NAME (5 mM) vs 2 times in the presence of D-NAME (5 mM). 5. beta cell responses to KCl (5 mM) and tolbutamide (0.185 mM) were only slight increased by L-NAME (5 mM) to values not far from the sum of the effects of L-NAME and of the two drugs alone. D-NAME (5 mM) was totally ineffective on the actions of both secretagogues. 6. L-NAME, infused 15 min before and during a rise in glucose concentration from 5 to 11 mM, was able in the low millimolar range (0.1-0.5 mM) to blunt the classical biphasic pattern of beta cell response to glucose and, at 5 mM, to convert it into a significantly greater monophasic one. In contrast, D-NAME (5 mM) was unable to induce similar effects. 7. SNP alone at 3 microM was ineffective but at 30 microM substantially reduced to second phase of insulin response to glucose; however, at both concentrations the NO donor partly reversed alterations in insulin secretion caused by L-NAME (5 mM) and restored a biphasic response.     

Impairment of insulin secretion in man by nifedipine

Summary The effect of nifedipine, a calcium antagonist, on carbohydrate metabolism and insulin secretion was evaluated in patients who required treatment with this drug. 20 subjects underwent two oral glucose tolerance tests (100 g), one under basal conditions, and the other after ten days of treatment with nifedipine 30 mg/day by mouth, in three divided doses. 10 subjects had normal glucose tolerance; in them nifedipine administration reduced the insulin response to oral glucose in the first 60 min, but improved glucose tolerance. The other 10 subjects had impaired glucose tolerance and nifedipine treatment resulted in a further reduction both of insulin secretion and glucose tolerance. No such effects were seen in the placebo (weight- and disease-matched) group. The mechanism by which nifedipine influences carbohydrate metabolism and insulin secretion is discussed.     

The dihydropyridine derivative,Bay K 8644, enhances insulin secretion by isolated pancreatic islets

Summary The effects of the dihydropyridine derivative Bay K 8644 upon insulin secretion by perifused isolated mouse pancreatic islets were examined. At a non-stimulatory glucose concentration (5 mmol/l) Bay K 8644 (1 mol/l) did not stimulate insulin release. However, the same drug concentration enhanced the insulin secretory responses to an intermediate (15 mmol/l) or high (30 mmol/l) glucose concentration by 80 or 90%, respectively. Bay K 8644 was half maximally effective at 0.1 mol/l and maximally effective at 1 mol/l. The results are compatible with the view that voltage-dependent calcium channels are essential for stimulus-secretion coupling in pancreatic B-cells.Part of the medical thesis of M.-T. Schrader     

Effects of L-dopa-induced dopamine accumulation on 45Ca2+ efflux and insulin secretion in isolated rat islets

It has previously been demonstrated in several species that the secretory granules of pancreatic beta-cells have the ability to store substantial amounts of calcium and bioactive amines, such as dopamine and serotonin. Furthermore, evidence for a similar topographical localization for amine and calcium within the periphery of the granules has been obtained. In the present study, a possible interaction between dopamine and calcium on insulin release was investigated. Isolated rat islets were loaded with 45Ca2+ in the presence of theophylline and high glucose and then perifused in a dynamic system where radioactivity and insulin were determined in the effluent. When perifused in a bicarbonate buffer with 2 mmol/l Ca2+ and supplemented with the monoamine oxidase inhibitor pargyline, L-3,4-dihydroxyphenylalanine (L-dopa)-induced dopamine accumulation in the islets brought about a slight and transient increase in 45Ca2+ efflux. This increase was more pronounced and sustained in a Ca2+-deficient buffer or in a Ca2+-deficient buffer supplemented with ethyleneglycolbis(aminoethylether)tetraacetic acid (EGTA). Insulin release was transiently stimulated by islet dopamine accumulation in the Ca2+-deprived media, but not in a medium with 2 mmol/l Ca2+. Glucose-induced insulin release in 2 mmol/l Ca2+ was potentiated by acute dopamine accumulation. The combined effect of glucose stimulation and islet accumulation of dopamine induced a transient insulin release in the Ca2+-deprived media with and without EGTA. This release of insulin was accompanied by an increased 45Ca2+ efflux which was most pronounced in the presence of EGTA. Stimulation with glucose alone, i.e. without addition of L-dopa tended to decrease insulin release and 45Ca2+ efflux in a Ca2+-deficient medium. No effects of L-dopa or L-dopa + glucose were encountered in a Ca2+-deficient buffer when the monoamine oxidase inhibitor pargyline was replaced by the dopa-decarboxylase inhibitor benserazide. The results are interpreted as being a consequence of a complex interaction between the accumulated dopamine and a pool of Ca2+ mainly confined to the secretory granules. This interaction could be followed by a transient increase in cytosolic Ca2+ and a subsequent efflux of Ca2+ out of the cell, eventually accompanied by insulin release. Increasing the cytosolic Ca2+ by acute dopamine accumulation makes the cell more sensitive to a concomitant stimulation with glucose, and the release of insulin is triggered. A long-term dopamine accumulation. On the other hand, might diminish the granular Ca2+ pool to such a level where insulin release is inhibited after stimulation with certain secretagogues.     

Autoradiographic localization of apamin-sensitive Ca2+-dependent K+ channels in rat brain

Endogenous opiates have been suggested to have effects on insulin secretion. In order to investigate the in vivo importance of endogenous opiate receptor agonists on basal and stimulated insulin secretion, the effect on plasma insulin of opiate receptor blockade by naloxone was studied in the mouse. It was found that the plasma insulin levels in naloxone-injected mice were moderately lower than in the control animals after 5 min. Ten minutes later, however, naloxone-injected animals had slightly higher plasma insulin levels than did the controls. Naloxone moderately potentiated the insulin secretory responses to glucose and the β₂-adrenoceptor agonist terbutaline. On the contrary, naloxone had no apparent effect on the insulin secretory response to the cholinergic agonist carbachol or the synthetic C-terminal octapeptide of cholecystokinin, CCK-8. In conclusion, endogenous opioid substances might moderate the regulation of basal insulin levels in the mouse. Further, they seem to have the capability to modulate the insulin release stimulated by glucose or β-adrenoceptor agonists.     

The in vivo interaction between gliclazide and glibenclamide and insulin on glucose disposal in the rat.

Many reports suggest that extrapancreatic actions contribute to the antidiabetic effect of sulphonylurea drugs (SUs). In this work, the ability of two SUs, namely, gliclazide and glibenclamide, to augment insulin action was studied in vivo. Both drugs elevated the plasma concentration of immunoreactive insulin (IRI) and lowered the plasma concentrations of glucose and non-esterified fatty acids (NEFA) in normal intact rats. These changes were not reproduced in alloxan-diabetic or eviscerated rats. The actions of insulin on plasma glucose and NEFA were not augmented by gliclazide in alloxan-diabetic rats. Neither gliclazide nor glibenclamide (given acutely and for 30 days) augmented the actions of exogenously administered insulin in reducing plasma glucose or NEFA concentrations in intact or eviscerated animals. It was concluded that these SUs do not produce their acute or chronic effects on blood glucose by augmenting the actions of insulin.     

Resveratrol enhances insulin secretion by blocking K(ATP) and K(V) channels of beta cells

The present study investigated the effect of resveratrol on the electrophysiology and insulin secretion of pancreatic beta cells, and examined resveratrol-induced alterations in insulin levels and plasma glucose of normal and streptozotocin-induced diabetic rats. Whole-cell voltage clamp study in the MIN6 cell, a mouse beta cell line, revealed that resveratrol significantly inhibited ATP-sensitive K(+) current at 3 micromol/l, and voltage-gated K(+) currents at 30 micromol/l. Ca(2+)-activated K(+) current was activated by resveratrol at 100 micromol/l. In MIN6 cells stained with membrane potential dye DiBAC(4)(5), resveratrol markedly depolarized membrane potential at the concentrations of 3-100 micromol/l. Insulin secretion was increased in the presence of resveratrol in MIN6, Hit-T15, and RIN-m5F cells. Resveratrol (3 mg/kg, i.p.) increased insulin secretion associated with a lowering in plasma glucose in normal rats, but not in streptozotocin-diabetic rats within the initial 60 min. In conclusion, resveratrol can act as an insulin-secretagogue through I(KATP) and I(KV) inhibition which can contribute to plasma glucose lowering effect in normal rats.     

大黄酸对db/db小鼠胰岛功能及炎症氧化损伤标志物表达的影响

目的通过先天性2型糖尿病动物模型db/db小鼠,探讨大黄酸对胰岛功能及炎症、氧化损伤标志物表达的影响。方法选取30只4周龄db/db雄性小鼠,随机分成治疗组和对照组,每组15只。治疗组每日固定时间给予大黄酸(120mg/kg,1%纤维素钠溶解)灌胃,对照组给予相同体积的1%纤维素钠,连续给药8周。投药结束后行经腹腔葡萄糖耐量试验(IPGTT)并测定胰岛素水平,用曲线下面积(AUC)代表胰岛素分泌水平,并通过计算IPGTT的0~30min胰岛素AUC评估早期胰岛素分泌功能。同时对小鼠胰腺进行胰岛素、核因子-κB(NF-κB)及8-羟基脱氧鸟苷(8-OHdG)免疫组织化学染色。结果与对照组相比,治疗组糖负荷后0,30,60,120min的血糖水平明显下降,而30,60,120min的胰岛素水平明显升高,尤其是早期相胰岛素水平升高更明显。同时,大黄酸治疗组小鼠胰岛素染色明显增强,NF-κB及8-OHdG表达明显受抑制。结论早期大黄酸治疗可以明显改善db/db小鼠的葡萄糖耐量,恢复早期胰岛素分泌功能,保护胰岛功能;同时早期大黄酸治疗明显减少炎症、氧化损伤标志物的表达。