Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.     

Impact of the two-layer method on the quality of isolated pancreatic islets

BACKGROUND/AIMS: We have already reported that the two-layer method (UW/PFC) reduces warm and cold ischemic injuries before islet isolation, and results in improvement of islet yield and viability. In this study, we try to evaluate the effect of the two-layer method on isolated islets. METHODOLOGY: We used male Wister rats. Isolated islets were cultured or preserved in various conditions for 24 hours. In group 1, islets were not cultured (control). In group 2, islets were cultured in RPMI at 37 degrees C. In groups 3 and 4, islets were cultured with "modified" two-layer method (RPMI/PFC) at 37 degrees C and 4 degrees C, respectively. In groups 5 and 6, islets were preserved in UW and with the two-layer method (UW/PFC), respectively at 4 degrees C. Islets in each group were evaluated in terms of function and viability in vitro. RESULTS: Stimulation Indices were 1.3, 2.6, 3.7, 1.2, 1.4, and 2.4 in groups 1, 2, 3, 4, 5 and 6, respectively. Islets in groups 2, 3 and 6 showed clear response to glucose stimulation. Among these 3 groups, the total viability of islets assessed by FDA/PI staining was 88%, 92%, and 76% in groups 2, 3, 6, respectively. CONCLUSIONS: Although in vivo studies are mandatory, the present study is supportive that the "modified" two-layer method (RPMI/PFC), which uses oxygenated PFC and RPMI, may be superior to conventional culture method with RPMI. This method may achieve further improvement of islet viability before implantation.     

Leptin inhibition of insulin secretion from isolated human islets

Leptin is a hormone produced and secreted from the adipose tissue. Its physiological actions include the regulation of satiety, food intake and energy balance. The production of leptin is increased by high insulin levels. Here, we demonstrate that leptin acts as an inhibitor of glucose-induced (20 mM) insulin secretion from isolated human islets. No effect was observed in the presence of lower glucose levels (2.8 and 10 mM glucose). The pancreatic β-cell might represent a target of a direct physiological action of leptin. We suggest the presence of an “adipo-insular axis” in which leptin mediates negative feedback from the adipose tissue to the endocrine pancreas. Received: 21 July 1997 / Accepted in revised form: 1 October 1997     

A method for enzymatic dissociation of cells from isolated pancreatic islets

Summary An enzymatic method for isolation of single cells from the islets of Langerhans is described. The isolated cells appeared well preserved and survived for at least 7 days when maintained in culture. The dry mass of the isolated islet cells was found to be decreased 30 min after administration of alloxan to obese-hyperglycemic mice. Isolated individual islet cells from obese-hyperglycemic mice had a higher dry mass than those from their lean litter mates. Traduzione a cura di G. U.     

Translation of messenger ribonucleic acid from isolated pancreatic islets and human insulinomas.

RNA preparations from isolated rat pancreatic islets and from human insulinomas were injected into oocytes of Xenopus laevis which were then incubated with [3H]leucine. Acid-ethanol extracts of the oocytes were immunoprecipitated with anti-insulin serum using the double antibody technique. Sodium dodecyl sulfate disc gel electrophoresis of the immunoprecipitates showed the presence of an insulin-displaceable immunoreactive material with a molecular weight of about 18,000 in extracts from oocytes injected with RNA of 9-11 S. This immunoreactive product was not detected in extracts from oocytes injected with buffer or 4-8S RNA or RNA heavier than 11 S. These observations suggest the involvement of a precursor larger than proinsulin in the biosynthesis of insulin.     

Immaturity of insulin secretion by pancreatic islets isolated from one human neonate

Human β‐cells are functionally mature by the age of 1 year. The timeline and mechanisms of this maturation are unknown owing to the exceptional availability of testable tissue. Here, we report the first in vitro study of insulin secretion by islets from a 5‐day‐old newborn. Glucose was inefficient alone, but induced insulin secretion, which was concentration‐dependent, showed a biphasic time‐course and was of similar magnitude as in infant islets when β‐cell cyclic adenosine monophosphate was raised by forskolin. Tolbutamide alone was effective in low glucose, but its effect was not augmented by high glucose. Metabolic amplification by glucose was thus inoperative, in contrast to amplification by cyclic adenosine monophosphate. Newborn islets showed high basal insulin secretion that could be inhibited by diazoxide or omission of CaCl₂. Postnatal acquisition of functional maturity by human β‐cells implicates control of basal secretion and production of metabolic signals able to activate both triggering and amplifying pathways of insulin secretion.     

Insulin inhibits its own secretion from isolated,perifused human pancreatic islets

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-m sized-islets) were sequentially perifused at 37°C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37°C culture in CMRL 1066 medium with or without the addition of either 200 or 400 U/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perufusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) U/ml added insulin, basal Cp release was 0.12±0.03, 0.14±0.02 and 0.14±0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47±0.13 ng/ml,P<0.02) and Ins400 (0.68±0.15 ng/ml,P<0.05) than C (0.83±0.15 ng/ml). The second-phase secretion peak was also significantly (P<0.05) reduced with added insulin (Ins200: 0.47±0.08 ng/ml; Ins400: 0.45±0.07 ng/ml) than in its absence (C: 0.65±0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6±2.3 ng/ml,P=0.03) and Ins400 (11.8±2.3 ng/ml) than with C (16.0±2.2 ng/ml). Thus, the addition for 24 h of either 200 or 400 U/ml insulin in the culture medium caused a significant decrease of insulin (as assessed by Cp measurement) secretion from perifused human islets, suggesting that feedback suppression of insulin release is at least in part due to a direct action of insulin on the islets.     

Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans

Aims: The role of cannabinoid receptors in human islets of Langerhans has not been investigated in any detail, so the current study examined CB1 and CB2 receptor expression by human islets and the effects of pharmacological cannabinoid receptor agonists and antagonists on insulin secretion. Methods: Human islets were isolated from pancreases retrieved from heart‐beating organ donors. Messenger RNAs encoding human CB1 and CB2 receptors were amplified from human islet RNA by RT‐PCR and receptor localization within islets was identified by immunohistochemistry. Dynamic insulin secretion from human islets perifused with buffers supplemented with CB1 and CB2 receptor agonists and antagonists was quantified by radioimmunoassay. Results: RT‐PCR showed that both CB1 and CB2 receptors are expressed by human islets and immunohistochemistry indicated that receptor expression co‐localized with insulin‐expressing β‐cells. Perifusion experiments using isolated human islets showed that insulin secretion was reversibly stimulated by both CB1 and CB2 receptor agonists, with CB1 receptor activation associated with increased basal secretion whereas CB2 receptors were coupled to initiation and potentiation of insulin secretion. Antagonists at CB1 (N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) and CB2 (N‐(1,3‐Benzodioxol‐5‐ylmethyl)‐1,2‐dihydro‐7‐methoxy‐2‐oxo‐8‐(pentyloxy)‐3‐quinoline carboxamide) receptors failed to inhibit the stimulatory effects of the respective agonists and, unexpectedly, reversibly stimulated insulin secretion. Conclusions: These data confirm the expression of CB1 and CB2 receptors by human islets and indicate that both receptor subtypes are coupled to the stimulation of insulin secretion. They also implicate involvement of CB1/2 receptor‐independent pathways in the antagonist‐induced stimulatory effects.     

血管紧张素Ⅱ与人离体胰岛功能研究

目的探讨分离纯化的人胰岛表面血管紧张素Ⅱ1型受体(AT1)的存在与否,了解血管紧张素Ⅱ对人胰岛分泌胰岛素功能的影响。方法对分离纯化后的胰岛进行AT1受体和胰岛素的免疫荧光双标法检测;使用不同剂量血管紧张素Ⅱ探讨其对人胰岛胰岛素释放反应的影响。结果免疫荧光双标记染色胰岛AT1受体和胰岛素均呈强阳性;血管紧张素Ⅱ对高糖刺激胰岛素分泌具有剂量依赖的抑制效应,用AT1受体拮抗剂预处理胰岛能拮抗这种抑制作用。结论分离纯化的人胰岛存在AT1受体,血管紧张素Ⅱ能够通过AT1受体直接抑制胰岛的胰岛素释放反应。     

Mebendazole and insulin secretion from isolated rat islets

In a preliminary communication we reported that mebendazole, a vermicide, decreased plasma glucose and free fatty acid concentrations and increased plasma C peptide concentrations in both type II diabetic patients. Therefore, we suggested that mebendazole was an insulin secretagogue. However, these were uncontrolled studies, and improved metabolic control in these patients due to spontaneous remission rather than drug-induced insulin secretion was a possibility. To investigate the direct effect of mebendazole on insulin secretion we used intact islets isolated from normal rat pancreata. Mebendazole in concentrations as low as 10 to 20 mumol/L caused a twofold to threefold increase in acute-phase insulin release from isolated perifused rat islets. This heightened insulin release occurred in the presence of glucose-stimulated insulin secretion.     

Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two-step separation procedure

Cord blood (CB) transplantation is primarily performed in children, rather than in adults, due to the low number of haemopoietic progenitor cells obtained from the small volume of a single CB collection. Prolonged thrombocytopenia is a major problem following CB transplantation. Efforts are currently underway to expand the number of CB progenitor cells ex vivo , in order to enable transplantation in adults and to decrease the period of thrombocytopenia. In this study we investigated different techniques for enrichment and expansion of megakaryocyte (Mk) progenitor cells and haemopoietic stem cells from CB. CBs from 20 normal deliveries were depleted of red blood cells (RBC) by dividing each sample and testing cell separation on 3% gelatin, Hespan, Ficoll-Paque or a two-step 3% gelatin followed by Ficoll-Paque separation. The two-step procedure was found to be superior to the other methods in enrichment of the Mk progenitor cells (CFU-Mk) (34.3-fold), while at the same time retaining the number of myeloid and erythroid progenitors, CD34⁺ and CD41⁺ cells. In short-term (14 d) liquid culture of non-adherent nucleated cells isolated by gelatin and Ficoll-Paque, a 40-fold expansion of clonable Mk progenitor cells was obtained in the presence of thrombopoietin (r-hu-TPO) and stem cell factor (r-hu-SCF). In similar cultures of isolated CD34⁺ cells, a 100-fold clonable Mk progenitor was obtained at day 14. Therefore this new technique may facilitate the ex vivo expansion of Mk progenitor cells and be adopted for future use in CB transplantation.     

Autocrine insulin action activates Akt and increases survival of isolated human islets

Aims/hypothesis The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt.Methods In order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period.Results We demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation.Conclusions/interpretation Our findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.     

Fluorescence of oxidized flavoproteins from perifused isolated pancreatic islets

In perifused pancreatic islets, the fluorescence of oxidized flavoproteins (FAD) was recorded continuously. Elevation of glucose concentration in the medium form 0 or 5 mM to 20 mM led to decrease in FAD-fluorescence beginning 10 sec after change of medium. L-leucine (10 mM), (+/-)-B-BCH (20 mM) and alpha-ketoisocaproic acid (10 mM) caused typical kinetics of FAD-fluorescence decrease. The results are interpreted to indicate rapid changes of the functional state of B-cell mitochondria induced by the above-mentioned stimulators of insulin release.     

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.     

Affinity-purified human Interleukin I is cytotoxic to isolated islets of Langerhans

Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play an important role in the molecular mechanisms underlying autoimmune B-cell destruction leading to Type 1 (insulin-dependent) diabetes mellitus.     

Somatostatin-induced inhibition of insulin secretion by isolated pancreatic rat islets prepared by micro-dissection or collagenase digestion.

The effect of somatostatin on insulin secretion in response to a variety of stimuli was investigated in micro-dissected as well as collagenase isolated pancreatic rat islets. Somatostatin inhibited insulin secretion in both islet preparations in the presence of different initiators, but failed to affect the hormone output induced by theophylline, 1-methyl-3-isobutylxanthin (IBMX) or p-chloromercuriphenylsulfonic acid (CMBS). The inhibitory action was associated with decreased glucose metabolism by islets (measured as conversion of U-14C-glucose to 14CO2).