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Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets 总被引:15,自引:0,他引:15
Summary Islet amyloid polypeptide is a novel 37 amino-acid-residues polypeptide which has been isolated from amyloid deposits in an insulinoma, and in human and cat islets of Langerhans. The molecule has 46% homology with the calcitonin gene-related peptide. Light microscopy examination of the pancreas shows that islet amyloid polypeptide immunoreactivity is restricted to the islet B cells. The present study utilized a rabbit antiserum against a synthetic peptide corresponding to positions 20–29 of islet amyloid polypeptide, a sequence without any amino-acid identity with calcitonin gene-related peptide. By applying the immunogold technique at the ultrastructural level, it was shown that both insulin and islet amyloid polypeptide immunoreactivity occurs in the central granular core of the human B cell secretory granules, while the A cells remain unlabelled. The demonstration that islet amyloid polypeptide is a granular protein of the B cells may indicate that it is released together with insulin. Further studies are necessary to evaluate the functional role of islet amyloid polypeptide. 相似文献
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A method for the recognition and separation of human blood monocytes on density gradients 总被引:7,自引:1,他引:6
Loos H; Blok-Schut B; van Doorn R; Hoksbergen R; Brutel de la Riviere A; Meerhof L 《Blood》1976,48(5):731-742
The density distribution of human mononuclear blood leukocytes was studied in order to define the optimal conditions for the separation of monocytes and lymphocytes by isopycnic centrifugation. Under standardized conditions, two populations of cells with partially overlapping, normally distributed densities were consistently found. The cells with the lowest density were recognized as monocytes, using phagocytosis and size distribution analysis as criteria. Since the density of monocytes continuously increased during the centrifugation, optimal separation of monocytes and lymphocytes could only be achieved by limiting the time of centrifugation to 10 min at 2200 g and 4 degrees C. The separation on discontinuous density gradients decreased when the load exceeded 8 X 10(6) mononuclear cells per sq cm. Analysis of the composition of the two cell populations obtained after separation on a three-layer discontinuous gradient revealed that the contamination of the monocytes with lymphocytes was due to the partial overlapping density distributions of both cell types. A small and a large scale method for isolation of monocytes from blood on discontinuous density gradients are presented. Under the described conditions, a preparation of functionally intact monocytes can be obtained which is comparable, both in yield and purity, to those obtained by methods based on surface adherence without the drawbacks of the latter methods. 相似文献
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We studied the contribution of the constitutive and the regulated pathways to the total secretion of islet amyloid polypeptide (IAPP) in human pancreatic islets after prolonged culture at either 5.5 or 24.4 mM glucose. In islets cultured in low concentrations of glucose, the secretion of IAPP in response to glucose was unaffected by brefeldin A (BFA) and completely blocked by ethyleneglycoltetraacetic acid. In islets cultured in high glucose concentrations, it was strongly inhibited by both agents. BFA had no effect on the glucose-induced insulin secretion. The determination of the islet peptide contents and the mRNA levels revealed a several-fold increase in the IAPP/insulin molar ratio of islets cultured in high glucose concentrations. Thus, prolonged exposure of human islets to high concentrations of glucose results in an increase in the synthesis of IAPP with respect to insulin. As a result, the release of IAPP through a mechanism sensitive to BFA is favored. These data support the hypothesis that IAPP and insulin are regulated in a noncoordinated way in human pancreatic islets. 相似文献
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目的观察硝基酪氨酸(NT)在糖尿病大鼠胰岛中的表达,以及普罗布考的干预对其表达和对胰岛β细胞的影响。方法大鼠高脂高糖饲料喂养4周后,腹腔注射链脲佐菌素30mg/kg复制2型糖尿病大鼠模型,普罗布考组(PB组)每天同时灌胃普罗布考500mg/kg,10周后测定空腹血糖(FPG)、空腹胰岛素(Fins)、总胆固醇(TC)、甘油三酯(TG)、丙二醛(MDA)、超氧化物歧化酶(SOD),计算胰岛素敏感指数(ISI),用免疫组化的方法观察NT在胰岛中的表达。结果糖尿病组(DM组)及PB组大鼠的FPG、TG、TC及MDA水平较正常对照组(NC组)明显增加(P〈0.01),NT的平均光密度也明显高于NC组(P〈0.01),但PB组上述指标的测定显著低于DM组(P〈0.01);DM组和PB组大鼠的SOD水平、ISI明显低于NC组(P〈0.01),PB组这些指标的测定显著高于DM组(P〈0.01);NC组和PB组的Fins比较差异没有统计学意义,但都明显高于DM组(P〈0.01)。结论NT在胰岛的表达增强,普罗布考作为抗氧化剂,对于STZ引起的或糖尿病引起的氧化应激,均在一定程度上减轻其损伤,保护了胰岛功能。 相似文献
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The major indication for pancreas or islet transplantation is diabetes mellitus type I. This process has to supply the insulin necessity keeping glucose under control. We have studied isogenic islet transplantation on the rat (WAG-RT1u) liver. The method of isolation and purification of the islets obtained 2.834 +/- 551.64 islets with purity of 83 +/- 2.45%. Diabetes was induced by streptozotocin and seric glucose prior transplantation was 35 mmol/L. The islet transplantation of 2.834 +/- 551.64 islets in the rat liver has normalized glucose test from 9.62 +/- 2.65 mmol/L 10 days after transplantation to 7.43 +/- 0.27 mmol/L later in the follow-up (P < 0.05). The median survival time of the islets was 73 days. In conclusion both the method of isolation and purification of the islets and islet transplantation was effective in the control of the diabetes induced by streptozotocin with median survival time of both islet and rat more than 73 days when rats were sacrified. 相似文献
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Following digestion of a single pancreas from 30- to 50-day-old Sprague Dawley rats, many single cells and small cell clusters were obtained. Over a succeeding 5-day interval in tissue culture, many islet-like cell clusters were noted. They were smaller than isolated intact rat islets and appeared to develop from selective aggregation of islet cells. More than 1,000 of these islet-like clusters were obtained from a single pancreas. The physiologic characteristics of these cells indicated preservation of insulin secretory responses to glucose (30, 50, 100, 150, 200, and 300 mg/dl), theophylline (1.5 mM), and arginine (10 mM). This appears to be a useful method to follow when large quantities of islet cells are required, as for transplantation. 相似文献
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目的初步研究成人胰岛的分离、纯化方法,为同种异体胰岛移植治疗1型糖尿病进行前期准备。方法采用改良的Ricordi技术消化成人尸体胰腺,然后用连续性密度梯度离心法纯化胰岛。胰岛收获量以国际标准的胰岛当量(islet equivalent,IEQ)表示。结果完成10例成人胰岛分离和纯化,其中5例完成了胰岛当量的统计,胰岛收获量为6367~108725IEQ/胰腺,平均为47678.8IEQ/胰腺,平均每克组织收获2055IEQ。结论采用改进的人胰岛分离方法,可以获得较大产量的有活性的胰岛。 相似文献
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D. R. Chadwick G. S. M. Robertson H. Contractor S. Swift S. Rose S. T. Thirdborough R. Chamberlain R. F. L. James P. R. F. Bell N. J. M. London 《Acta diabetologica》1993,30(1):57-59
Euro-Ficoll (EF) and bovine serum albumin (BSA) are the two most commonly used media for the density gradient purification of human pancreatic islets. The aim of this study was to compare these two media with respect to the efficiency of human islet isolation. Ten human pancreata were collagenase-digested, and samples of digest were separated on either a continuous linear density gradient of BSA or a discontinuous gradient of EF (1.108/1.096/1.037/Euro-Collins). Efficiency of islet purification was assessed by insulin and amylase assay of aliquots aspirated from the BSA gradients, and from the interfaces of the EF gradients. Islets were obtained from two interfaces in the EF gradients. Islet yield from the upper interface was generally poor (median 28% of total insulin; range 2–71%), but purity was better than for an equivalent yield using BSA [1% (0–3%) amylase contamination for EF versus 6% (0–37%) for BSA;P=0.013]. Pooling both EF interfaces increased yield to 66% (17–81%) but markedly reduced purity [46% (0–50%) amylase for EF versus 31% (0–52%) for BSA]. In conclusion, the efficiency of human islet purification is similar, though disappointingly low, with BSA and with EF. Considerable scope exists, therefore, for improvement in the density gradient purification of human islets. 相似文献
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Insulin secretory actions of extracts of Asparagus racemosus root in perfused pancreas, isolated islets and clonal pancreatic beta-cells 总被引:2,自引:0,他引:2
Hannan JM Marenah L Ali L Rokeya B Flatt PR Abdel-Wahab YH 《The Journal of endocrinology》2007,192(1):159-168
Asparagus racemosus root has previously been reported to reduce blood glucose in rats and rabbits. In the present study, the effects of the ethanol extract and five partition fractions of the root of A. racemosus were evaluated on insulin secretion together with exploration of their mechanisms of action. The ethanol extract and each of the hexane, chloroform and ethyl acetate partition fractions concentration-dependently stimulated insulin secretion in isolated perfused rat pancreas, isolated rat islet cells and clonal beta-cells. The stimulatory effects of the ethanol extract, hexane, chloroform and ethyl acetate partition fractions were potentiated by glucose, 3-isobutyl-1-methyl xanthine IBMX, tolbutamide and depolarizing concentration of KCl. Inhibition of A. racemosus-induced insulin release was observed with diazoxide and verapamil. Ethanol extract and five fractions increased intracellular Ca(2+), consistent with the observed abolition of insulin secretory effects under Ca(2+) -free conditions. These findings reveal that constituents of A. racemosus root extracts have wide-ranging stimulatory effects on physiological insulinotropic pathways. Future work assessing the use of this plant as a source of active components may provide new opportunities for diabetes therapy. 相似文献
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Thyroxine treatment induced experimental hyperthyroidism in ob/ob mice, inhibited glucose-induced insulin secretion from the isolated perfused ob/ob mouse pancreas, and reduced total pancreas insulin content. In contrast, glucose-induced insulin release from incubated pancreatic islets and insulin content of pancreatic islets from ob/ob mice isolated by freehand microdissection were not reduced after thyroxine treatment when expressed per microgram dry islet. Histological examination of the ob/ob mouse pancreas revealed islets without degenerative lesions of islet cells. Granularity of beta cells was well preserved. The average number of pancreatic islets was unchanged. However, the beta cell area was significantly decreased in relation to the total pancreatic parenchyma after thyroxine treatment. This implies that insulin release and content per pancreatic islet was half of that of the controls. ATP content of islets was slightly reduced. Glucose oxidation and glucose utilization by islets from treated mice were slightly increased. Thyroxine treatment of the animals did not abolish the stimulation of 45Ca2+ uptake by glucose, but it did suppress the potentiating effect of fasting on the stimulatory effect of glucose on 45Ca2+ uptake. The metabolic characteristics of islets from experimentally hyperthyroid mice are those of all hyperthyroid tissues. The results provide no evidence for the view that the effects of thyroxine treatment may be due to disturbed metabolic function or energy deprivation of pancreatic islets. Inhibition of insulin secretion from the pancreas after thyroxine administration is apparently due to a reduction in pancreas insulin content and a diminished pancreatic islet volume. Reduced pancreatic islet volume represents most probably a reduction of individual islet cell volume. 相似文献
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Supplementation of islet culture medium with insulin may have a beneficial effect on islet secretory function 总被引:2,自引:0,他引:2
Recent reports suggest that apoptosis resulting from the disruption of the normal cell-matrix relationship (anoikis) during islet isolation could lead to a loss of islet tissue in culture. Insulin is known to have a role in cell growth and survival, and this study was undertaken to assess any beneficial effect on islets by supplementing the islet culture medium with insulin. Human and porcine islets were cultured in medium supplemented with 0, 10, 100, and 1,000 ng x mL(-1) insulin. Secretory function was assessed by perifusion at days 1 and 8. The results demonstrated a significant variation in stimulation index between isolations for human islets, but there was no effect relating to the concentration of insulin in the medium or time in culture. For porcine islets, there was a significant (p < 0.001) improvement in secretory function for islets cultured in 10 and 100 ng x mL(-1) insulin, relative to 0 and 1,000 ng x mL(-1) insulin. There was no interisolation variation or effect of time in culture. In conclusion, the secretory function of porcine islets benefited from the addition of 10 to 100 ng x mL(-1) insulin to the culture medium, but interisolation variation in human islet secretory function did not allow any specific effect of the insulin to be determined. 相似文献
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Summary Insulin responses to clinical grade human growth hormone (hGH), intact hGH, naturally occurring diabetogenic substance (NDS), and subtilisin cleaved forms of hGH (S1 and S3) were studied using hypophysectomized rat pancreatic islets. While clinical grade hGH (200 g/ml) elicited a prompt and sustained release of insulin, purified intact hGH (200 g/ml) did not. Naturally occurring diabetogenic substance, isolated from clinical grade hGH preparations, stimulated insulin release at 200 ng/ml. Upon repeat stimulation with NDS, a significantly greater insulin release than with initial stimulation was observed. Although S3 (200 /ml) elicited significant insulin release, S1 (200 g/ml) did not. Direct stimulation of insulin release with clinical grade hGH is not due to intact hGH but another protein(s) such as NDS. Enzymic modification of intact hGH appears to enhance insulin stimulatory capacity. 相似文献
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The use of continuous density gradients for the assessment of islet and exocrine tissue densities and islet purification 总被引:2,自引:0,他引:2
G. S. M. Robertson D. R. Chadwick H. Contractor R. F. L. James P. R. F. Bell N. J. M. London 《Acta diabetologica》1993,30(3):175-180
The purification of large numbers of human pancreatic islets remains one of the limiting factors in islet transplantation. This paper describes and validates a method for accurately and reproducibly determining the density of islets and exocrine tissue in pancreatic digest on the basis of their isopycnic distribution on linear continuous density gradients. The use of this data to analyse and compare the purity of a standard 60% islet yield is described. The results obtained using such gradients will enable factors responsible for the variation in yield between pancreases to be determined and optimized, improving the results and reliability of islet purification. 相似文献
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R Calafiore A Falorni M V Ambrosini G Basta M L Picchio P Brunetti 《Journal of endocrinological investigation》1991,14(8):629-633
A simple method for assaying DNA content of in vitro culture maintained pancreatic islets is described. By employing lyophilization of the islets and subsequent fluorometric analysis, we have been able to measure DNA from relatively small cell samples. The method may result advantageous, over others, previously reported, since it is less subject to experiment-related interfering conditions. This procedure, which is easy and reliable, even for very low DNA concentrations, seems to be very useful for culture maintained pancreatic islets, since in vitro work conducted with this endocrine tissue is usually associated with small size cell samples. The method may help for a simple assessment of the viable islet cell mass in in vitro studies. 相似文献
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Jansson L Bodin B Källskog O Andersson A 《European journal of endocrinology / European Federation of Endocrine Societies》2005,153(2):345-351
OBJECTIVES: The aim of this study was to evaluate islet blood-flow changes during stimulated growth of the islet organ without any associated functional impairment of islet function. DESIGN: A duct ligation encompassing the distal two-thirds of the pancreas was performed in adult, male Sprague-Dawley rats. METHODS: Pancreatic islet blood flow was measured in duct-ligated and sham-operated rats 1, 2 or 4 weeks after surgery. In some animals studied 4 weeks after surgery, islet blood flow was also measured also during hyperglycaemic conditions. RESULTS: A marked atrophy of the exocrine pancreas was seen in all duct-ligated rats. Blood glucose and serum insulin concentrations were normal. An increased islet mass was only seen 4 weeks after surgery. No differences in islet blood perfusion were noted at any time point after duct ligation. In both sham-operated and duct-ligated rats islet blood flow was increased during hyperglycaemia; the response was, however, slightly more pronounced in the duct-ligated part of the gland. CONCLUSIONS: Normal, physiological islet growth does not cause any major changes in the islet blood perfusion or its regulation. This is in contrast to findings during increased functional demands on the islets or during deteriorated islet function, when increased islet blood flow is consistently seen. 相似文献