Effect of pharmacological agents and fasting on pancreatic islet norepinephrine in the golden hamster

Summary Using a specific and sensitive radioenzymatic assay that utilizes the partially purified enzyme phenylethanolamine-N-methyltransferase, studies were done to determine if pharmacological agents and/or fasting alter the norepinephrine concentration of collagenase-isolated golden hamster pancreatic islets. The norepinephrine concentration (42.1±8.07 mol/kg net weight, mean±SEM) and the monoamine oxidase activity (5,407±530 pmol product /mg tissue/min) of hamster pancreatic islets was at least five times higher than acinar pancreas, kidney, heart, median eminence or cerebral cortex. The catechol-o-methyltransferase activity of hamster islets (7±2.3 pmol product/mg tissue/min) was one half or less than the other tissues. Islet norepinephrine was not increased by two days administration of the monoamine oxidase inhibitor tranylcypromine. Islet norepinephrine concentration was increased 2-fold by administration of the norepinephrine precursor DL-threo-dihydroxyphenylserine.This increase was enhanced by prior administration of tranylcypromine (3.5-fold) and prevented by prior administration of the decarboxylase inhibitor N¹-(DL-seryl)-N²-(2,3,4-trihydroxybenzyl) hydrazine (RO-4-4602). There was a good correlation between the islet norepinephrine concentration and the plasma glucose concentration after pharmacological agents. Reserpine administration markedly depleted the islet norepinephrine concentra tion. Fasting of 24, 48 and 72 h did not alter the norepinephrine concentration in islets and heart. It is concluded that the pancreatic islets of the hamster have an active noradrenergic system, but that islet norepinephrine does not appear to play an important role in the impaired insulin secretion of fasting hamsters.     

Intracellular pancreatic B cell serotonin and the dynamics of insulin release

Summary The role of intracellular pancreatic B cell serotonin in the dynamics of insulin release was investigated in an in situ perfused rat pancreas preparation. Animals were pretreated with 5-hydroxytryptophan (5-HTP) to increase the intracellular levels of serotonin (5-HT) as shown by fluorescence histochemistry. Despite a clear induction of intracellular 5-HT fluorescence in pancreatic islets neither the pattern nor the total amount of insulin released were significantly modified after perfusion with either glucose or tolbutamide. However, the L-amino acid decarboxylase inhibitor, RO 4-4602, significantly decreased both phases of glucose-mediated insulin release in normal animals as well as in those receiving 5-HTP.     

Monoamines in the pancreatic islets of the mouse

Summary By application of autoradiographic technique the cellular and subcellular distribution of radio-activity in mouse pancreatic islets was investigated following intravenous administration of³H-5-hydroxytryptophan. Autoradiographic silver grains, most of which probably represent 5-hydroxytryptamine formed from the labelled precursor, appeared over A₂ and B cells, whereas very few grains were recorded over A₁ cells at any time investigated (20 min–16 hours) and also when monoamine oxidase was inhibited. Quantitative analysis of autoradiographic sections revealed that the concentration of silver grains over the specific granules of A₂ and B cells was 5–10 times higher than over the remaining parts of these cells. In A₂ cells the highest grain count was recorded at 20 minutes, in B cells at 1 hour after the injection of label. After 8 hours very few, and after 16 hours no silver grains appeared over islet cells. Inhibition of monoamine oxidase caused an increased retention of label over islet cells, most pronounced over A₂ cells. Pretreatment with reserpine abolished the autoradiographic reaction.This study was supported by Grant K71-12X-3352-01 from the Swedish Medical Research Council.     

Inhibition of glucagon release by serotonin in mouse pancreatic islets

Summary In this work we have investigated the effect of serotonin on glucagon release in mouse pancreatic islets isolated by the collagenase technique.Incubation of the islets with serotonin (4 ×10^–3mol/l) was associated with an inhibition of glucagon output both in the basal medium (3.3 mmol/l glucose) and in the presence of arginine (10 mmol/l). The inhibitory effect of serotonin on basal glucagon release was also apparent at concentrations of 2×10^–3 mol/l, 10^–3 mol/l and 5×10^–4 mol/l. Addition of 5-hydroxytryptophan (4 ×10^–3 mol/l) to the incubation medium was without effect on basal glucagon output while it significantly reduced arginine-induced glucagon release. In contrast, tryptophan (4×10^–3 mol/l) provoked glucagon secretion. As inferred from our previous human studies, the present data indicate that serotonin is able to inhibit glucagon secretion. These findings provide further support for the participation of a serotoninergic mechanism in the control of A-cell function.     

Effects of Season, Pinealectomy, and Blinding, Alone and in Combination, on Hypothalamic Monoaminergic Activity in the Teleost Channa punctatus (Bloch)

In Channa punctatus, day-night variations in hypothalamic 5-HT (serotonin) and monoamine oxidase (MAO) activity were noticed in preparatory but not other phases (prespawning and postspawning) of the annual reproductive cycle. Hypothalamic MAO, 5-HT, and norepinephrine (NE) activity was found to be high in the prespawning phase and low in the postspawning phase. Dopamine (DA) activity, on the other hand, was high in the postspawning season and low in the prespawning phase. Pinealectomy caused season-dependent effects on hypothalamic monoaminergic activity, with a significant increase in serotonergic activity and a significant reduction in MAO activity at midscotophase during the preparatory phase (March) but not in the prespawning (May-June) or postspawning (September) phases. Hypothalamic catecholaminergic (CA) activity was not influenced by pinealectomy during any of the seasons. To determine whether or not the photoperiodic influences on daily variations of 5-HT and MAO in the preparatory phase are mediated via pineal and/or lateral eyes, fish were pinealectomized and/or blinded in January, when there is no rhythm, and sacrificed in February, when a day-night variation normally sets in. The day-night difference in 5-HT content and activity and MAO activity was not abolished by pinealectomy or blinding alone; but the combination (pinealectomy + blinding) obliterated the daily variation only in 5-HT content and in MAO activity. However, pinealectomy and blinding, alone or in combination, caused a significant elevation of 5-HT activity (not its level) and a significant decrease in MAO activity at midscotophase, with the combination having an additive effect. Hypothalamic CA content or activity was not affected by these regimes. The results show that photoperiodic influence on the daily pattern of 5-HT and MAO activity is mediated through and by the interaction of the pineal and lateral eyes.     

醛糖还原酶抑制剂对小鼠胰岛体外培养时胰岛素分泌的影响

观察了ＩＣＩ１２８４３６（Ｓｔａｔｉ），一种新型醛糖还原酶抑制剂（ＡＲＩ），使体外培养时小鼠胰岛山梨醇形成减少后，葡萄糖诱发快速与慢速相胰岛素释放的变化情况。培养４８或９６小时后，葡萄糖诱发快速或慢速相胰岛素释放，ＡＲＩ组（１０ｍｇ／Ｌ）与对照组胰岛比较均无显著性差异。说明ＡＲＩ虽可使胰岛山梨醇形成减低，但其对葡萄糖诱发胰岛素释放无明显影响。提示胰岛山梨醇可能无确切促进胰岛素分泌的作用。     

Effect of human lymphoblastoid interferon on insulin synthesis and secretion in isolated human pancreatic islets

Summary Human islets of Langerhans were isolated from the pancreas removed from a 13-year-old female transplant donor. The islets were incubated in a culture medium for 24 h in the presence of human lymphoblastoid interferon (1000 units/ml). Insulin secretion, proinsulin biosynthesis, total protein biosynthesis and total insulin content were assessed at various concentrations of glucose in the presence of interferon. In interferon-treated islets glucose-stimulated insulin secretion was unaltered from that of control islets; however, glucose-stimulated proinsulin biosynthesis was specifically inhibited by interferon (48%, p<0.025). Total protein biosynthesis and total insulin content were not significantly affected by interferon.     

The effect of serotonin on in vitro insulin secretion and biosynthesis in mice

Summary The effect of serotonin on insulin secretion and biosynthesis was studied using isolated islets of mice. Serotonin produced a small stimulatory effect on insulin secretion when glucose was present in the incubation medium at a low concentration. On the other hand, an inhibition of insulin secretion was obtained with serotonin when glucose in the medium reached 3.0 mg/ml concentration. No significant effect of serotonin was obtained on insulin biosynthesis, neither in the presence of low nor with a high glucose concentration. These results suggest that the effect of this monoamine on insulin secretion is not mediated via its effect on insulin biosynthesis.Supported by Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg, SFB 87 Ulm     

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

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

他汀类调脂药对大鼠胰岛胰岛素分泌的影响及机制研究

目的 观察不同他汀类药物对大鼠胰岛葡萄糖刺激的胰岛素分泌(GSIS)的抑制作用及机制.方法 新鲜分离或经24 h培养的胰岛均匀分为对照组、阿托伐他汀组、氟伐他汀组和普伐他汀组,对照组给予Kreb-Ringer碳酸氢盐缓冲液,他汀类药物组分别给予100μmol/L阿托伐他汀、氟伐他汀和普伐他汀,水浴30 min或过夜培养24 h.各组经2.8、5.5、11.1、16.7、25.0 mmol/L葡萄糖刺激后,37℃水浴法测定胰岛GSIS变化,生物化学发光法测定三磷酸腺苷(ATP)含量.结果 100μmol/L阿托伐他汀水浴30 min后,在16.7 mmol/L葡萄糖刺激下,与相应对照组比较,ATP含量[(9.54±1.64)pmol/胰岛比(12.33±1.89)pmol/胰岛]及胰岛素分泌(1.60±0.21比2.39±0.30)均下降(P＜0.05);100 μmol/L氟伐他汀过夜培养24 h后,在16.7 mmol/L葡萄糖刺激下,与相应对照组比较,ATP含量[(10.24±2.01)pmol/胰岛比(12.31±2.16)pmol/胰岛]及胰岛素分泌(3.12±0.32比4.17±0.37)也均下降(P＜0.05).结论 阿托伐他汀、氟伐他汀通过抑制胰岛ATP的生成而抑制GSIS,抑制程度与其脂溶性强弱有关.

Abstract：

Objective To evaluate the inhibitory effect of statins on glucose-stimulated insulin secretion (GSIS) of pancreatic islet in rat and to explore its mechanisms. Methods According to the average volume, freshly isolated or 24-hour cultured pancreatic islets were randomly divided into control group( incubated with Kreb-Ringer bicarbonate buffer), the atorvastatin group( incubated with 100 μ mol/L atorvastatin), the fluvastatin group (incubated with 100 μ mol/L fluvastatin)and the pravastatin group (incubated with 100 μ mol/L pravastatin). Stimulated by 2. 8,5. 5,11.1,16. 7 mmol/L and 25.0 mmol/L glucose respectively, the effect of 100 μ mol/L statins on ATP content and GSIS was compared in the four groups. GSIS was performed by the 37℃ bath incubation method and ATP content was measured by chemiluminescence method. Results Incubated with 100 μ mol/L atorvastatin for 30 minutes, in the presence of 16. 7 mmol/L glucose, the ATP content [(9. 54 ± 1. 64) pmol/islet vs ( 12. 33 ± 1.89) pmol/islet] and GSIS (1.60 ± 0. 21 vs 2. 39 ± 0. 30) were significantly reduced in comparison with the control group (P＜0. 05). Cultured with 100 μmol/L fluvastatin for 24 hours, the ATP content [( 10. 24 ±2.01 )pmol/islet vs (12. 31 ±2. 16) pmol/islet] and GSIS (3. 12 ± 0. 32 vs 4. 17 ±0. 37 ) were all significantly decreased at the higher glucose concentration of 16. 7 mmol/L ( P ＜ 0. 05). Conclusion Atorvastatin and fluvastatin may inhibit GSIS by decreasing ATP content in pancreatic islet and the inhibitory effect is related to the strength of its lipophilicity.     

Effect of 2-deoxyglucose on islet cell morphology and insulin secretion

Summary The effect of 2-deoxyglucose (2-DG) on islet cell morphology and portal vein insulin levels was studied. The B-cells did not show any alteration in their fine structure, except for an increase in dense bodies. Six hrs after 2-DG administration degranulation was observed in some of these cells. Insulin levels were low 45 min after 2-DG administration, in spite of the marked hyperglycemia, and slightly higher than normal after 6 hrs. The A-cells remained well granulated at all times after 2-DG injection. The D-cells showed signs of functional activity, such as microvilli formation, increased lamellar ergastoplasm and free ribosomes. The significance of these findings is discussed. Traduzione a cura di G.U.     

Effect of extracellular pH on insulin secretion and glucose metabolism in neonatal and adult rat pancreatic islets

Changes in extracellular pH are known to affect glucose-stimulated insulin secretion. In the present study, glucose metabolism in pancreatic islets cultured at different pHs was investigated. Also, for islet transplantation purposes, insulin secretion and glucose metabolism were compared in neonatal and adult islets at different pHs to determine which islet preparation is more tolerant to acidity and alkalinity. The results revealed a dependency of insulin secretion on the external pH in both neonatal and adult islets. Reduction of insulin secretion was observed at both the acidic and alkaline sides of pH 7.3. Glucose stimulated increases of insulin secretion in all cases. Similar results were obtained for ATP and pyruvate contents. Intracellular insulin increased with the increase of pH value. In contrast, calcium content decreased with the increase of pH. The results demonstrate that neonatal islets are more acid tolerant than adult islets. Both basal and glucose-stimulated insulin secretions, as well as other parameters of neonatal islets were significantly higher than those of adult islets in response to low pH. The differences under alkaline conditions were not significant but give an indication that neonatal islets are more tolerant to alkalinity than are adult islets. Received: 10 February 2001 / Accepted in revised form: 29 June 2001     

Expression of the gene for a membrane-bound fatty acid receptor in the pancreas and islet cell tumours in humans: evidence for GPR40 expression in pancreatic beta cells and implications for insulin secretion

Aims/hypothesis G protein-coupled receptor 40 (GPR40) is abundantly expressed in pancreatic beta cells in rodents, where it facilitates glucose-induced insulin secretion in response to mid- to long-chain fatty acids in vitro. However, GPR40 gene expression in humans has not been fully investigated, and little is known about the physiological and pathophysiological roles of GPR40 in humans. The aim of this study, therefore, was to examine GPR40 expression and its clinical implications in humans.Methods: GPR40 mRNA expression in the human pancreas, pancreatic islets and islet cell tumours was analysed using TaqMan PCR.Results: GPR40 mRNA was detected in all human pancreases collected intraoperatively. It was enriched approximately 20-fold in isolated islets freshly prepared from the pancreases of the same individuals. The estimated mRNA copy number for the GPR40 gene in pancreatic islets was comparable to those for genes encoding sulfonylurea receptor 1, glucagon-like peptide 1 receptor and somatostatin receptors, all of which are known to be expressed abundantly in the human pancreatic islet. A large amount of GPR40 mRNA was detected in insulinoma tissues, whereas mRNA expression was undetectable in glucagonoma or gastrinoma. The GPR40 mRNA level in the pancreas correlated with the insulinogenic index, which reflects beta cell function (r=0.82, p=0.044), but not with glucose levels during the OGTT, the insulin area under the OGTT curve or the index for the homeostasis model assessment of insulin resistance (HOMA-IR).Conclusions/interpretation The present study provides evidence for GPR40 gene expression in pancreatic beta cells and implicates GPR40 in insulin secretion in humans.     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

分离SD大鼠胰岛接种于24孔板中,用不同浓度的葡萄糖和白藜芦醇分别培养1 h或24 h,结果表明白藜芦醇孵育大鼠胰岛1 h可呈剂苗依赖地抑制大鼠高糖刺激的胰岛素分泌,1、10和100 μmol/L白藜芦醇可以分别使胰岛素的分泌降低10%、35%(P<0.05)和80%(P<0.01).显微离子成像技术爪10μmol/L的白藜芦醇可以使高糖引起的β细胞内Ca2+浓度的升高减少60%(P<0.05).白藜芦醇可使软脂酸孵育24 h大鼠胰岛的胰岛素分泌恢复到对照组的75%(P<0.01),提示白藜芦醇短期可通过调控细胞内的Ca+浓度,而抑制原代胰岛高精刺激的胰岛素分泌,长期可改善软脂酸引起的β细胞损伤.     

The effect of biguanides on secretion and biosynthesis of insulin in isolated pancreatic islets of rats

Summary Based on the clinical observation that biguanide treatment of obese patients may alter insulin levels, the influence of metformin and phenformin on basal and glucose stimulated insulin secretion, as well as on insulin biosynthesis, was studied in isolated islets of rats. — Biguanide concentrations of 100 g/ml, or higher, significantly reduced glucose stimulated insulin secretion. Both dose dependence and a difference in the intrinsic activities of metformin and phenformin were demonstrated. Incubating the same islets for a second period without biguanides, glucose stimulated insulin secretion was still decreased. Addition of glibenclamide during this second period increased insulin secretion, but did not overcome complete inhibition achieved after incubation at very high biguanide concentrations. Glucose stimulated biosynthesis of proinsulin and insulin was decreased in the presence of biguanides and completely suppressed at very high concentrations. Inhibition of cell respiration in the islet cells effected by high biguanide doses may be the reason for the inhibition of secretion and biosynthesis of insulin. — On the other hand, an insulin release was found at the highest phenformin concentration of 10 mg/ ml and during perfusion of the isolated rat pancreas with higher biguanide doses. — Biguanide concentrations found to be effective in this study are very high compared with therapeutic levels. Moreover, biguanide actions are known to be highly dependent on species, concentration and metabolic situation. — Definite conclusions from these findings regarding clinical significance, therefore, seem unwarranted.Supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Effect of adrenalectomy on the development of a pancreatic islet lesion in fa/fa rats

Summary Adrenalectomy prevents development of obesity and hyperinsulinaemia in obese (fa/fa) Zucker rats, thereby implicating the hypothalamopituitary-adrenal axis in the pathogenesis of obesity. In this study glucose-induced insulin secretion and glucokinase activity were investigated in isolated islets from adrenalectomized and control obese and lean female rats. Islets from control fa/fa rats were more sensitive to glucose with a half-maximal effective concentration (EC₅₀) of 6.1±2.0 mmol · l^–1 compared with 10.6±2.7 mmol · l^–1 for adrenalectomized fa/fa rat islets. Adrenalectomy did not alter the islet sensitivity to glucose in the lean rats (EC₅₀ of 9.4±1.5 mmol · l^–1 and 9.3±2.0 mmol · l^–1 for adrenalectomized and control lean rats respectively). Mannoheptulose did not inhibit insulin secretion from control obese rats; however at concentrations of 1.0 mmol · l^–1 or more it significantly inhibited glucose-induced insulin secretion in adrenalectomized obese and lean, and control lean rat islets (p<0.05). In adrenalectomized fa/fa islets the glucokinase K_m was increased twofold compared with the control fa/fa rats (9.5±1.5 mmol · l^–1 vs 5.0±1.5 mmol · l^–1, respectively), but there was no significant change in glucokinase K_m in the lean rat islets after adrenalectomy. Mannoheptulose (10 mmol · l^–1) caused a significant reduction in glucose phosphorylation in disrupted islets of adrenalectomized fa/fa and lean, and of control lean rats, but not of control fa/fa rats. These data demonstrate that development of abnormal regulation of glycolysis in pancreatic islet beta cells of fa/ fa rats, as indicated by the insulin response to mannoheptulose and glucokinase activity, is dependent on an intact hypothalamo-pituitary-adrenal axis.Abbreviations ADX Adrenalectomy/adrenalectomized - CRH corticotrophin releasing hormone - DMEM Dulbecco's modified Eagle's medium - EC₅₀ half-maximal effective concentration - HPA hypothalamo-pituitary-adrenal - MH mannoheptulose - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid     

Effect of diacylglycerol lipase inhibitor RHC 80267 on pancreatic mouse islet metabolism and insulin secretion

Summary The effect of interference with diacylglycerol metabolism was investigated in pancreatic mouse islets. In the presence of the diacylglycerol lipase inhibitor RHC 80267, glucose-induced insulin secretion was reduced 50–60%; whereas carbacholin-induced insulin secretion was unaffected. Addition of the diacylglycerol kinase inhibitor R 59022 did not change glucose-stimulated insulin secretion but abolished the inhibition seen in the presence of RHC 80267. RHC 80267 increased islet glucose utilisation, measured as formation of tritiated water from 5-[³H]-glucose, 3-fold but did not affect glucose oxidation to CO₂, lactate production or islet ATP levels. Glucose utilisation in leucocytes and hepatocytes was not increased by addition of RHC 80267. Islet lipid production from glucose was augmented 4-fold in the presence of RHC 80267 but only accounted for about 5% of the increase in glucose utilisation. The activity of adenylate cyclase and phosphoinositide-specific phospholipase C was unaffected by RHC 80267. Concentrations of RHC 80267 below 35 mol/l did not alter the activity of phospholipase A₂; whereas higher concentrations of the drug inhibited phospholipase A₂ activity approx 25%. The data support the hypothesis that production of arachidonic acid from diacylglycerol may be involved in regulation of insulin secretion.Abbreviations RHC 80267 (1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane) - R 59022 6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-]pyrimidin-5-one     

Inhibition of insulin secretion,but normal peripheral insulin sensitivity,in a patient with a malignant endocrine pancreatic tumour producing high amounts of an islet amyloid polypeptide-like molecule

Summary Islet amyloid polypeptide or amylin is a polypeptide secreted mainly from the pancreatic beta cells together with insulin upon stimulation. High levels of islet amyloid polypeptide have also been shown to increase the peripheral insulin resistance and consequently a role for islet amyloid polypeptide in the glucose homeostasis has been suggested. We have studied the glucose homeostasis in a patient with a malignant endocrine pancreatic tumour producing large amounts of an islet amyloid polypeptide-like molecule (about 400 times the upper reference level for islet amyloid polypeptide). This patient developed insulin-requiring diabetes mellitus shortly after the tumour diagnosis. Both intravenous and oral glucose tolerance tests revealed inhibited early responses in insulin and C-peptide release, but the insulin and C-peptide response to glucagon stimulation was less affected. Aneuglycaemic insulin clamp showed normal insulin-mediated glucose disposal. In vitro experiments, where isolated rat pancreatic islets were cultured with serum from the patient, showed a moderately decreased islet glucose oxidation rate and glucose-stimulated insulin release compared to islets cultured with serum from healthy subjects. However, culture of rat islets with normal human serum supplemented with synthetic rat islet amyloid polypeptide did not affect the glucose-stimulated insulin release. In conclusion, the observed effects show that the diabetic state in this patient was associated with an impaired glucose-stimulated insulin release but not with an increased peripheral insulin resistance. Thus, the results suggest that if islet amyloid polypeptide has diabetogenic effects they are more likely to be exerted at the level of insulin secretion than at the level of peripheral insulin sensitivity.