Survival of cryopreserved isolated adult human pancreatic islets of Langerhans

Human islets of Langerhans were isolated from the pancreata of 13 adult organ donors, cultured for 24 h, cryopreserved, stored in liquid nitrogen at -196 degrees C for 6-88 days, thawed, and then cultured again. The number of islets recovered after this procedure was 80% of that present at the beginning. The viability of cultured/cryopreserved islets was then compared with that of islets from the same donor cultured for 24 hr, and was assessed by supravital staining, insulin response to glucose, and survival after implantation under the kidney capsule of nude rats. Supravital staining showed more nonviable cells in cryopreserved islets than in their cultured counterparts. Significant response to glucose was seen before and after cryopreservation in 2 of 4 sets of islets. Xenografts of 200 cultured islets (from 13 donors) were implanted in 15 nude rats under the kidney capsule, and a further 15 rats had cryopreserved islets (from the same 13 donors) similarly implanted beneath the kidney capsule. Two weeks later tissue was visible at the site of implantation in all 30 rats. Histological examination of both groups showed the tissue to have the morphology of islets, confirmed by immunohistochemical chemical localization of insulin. The insulin content of kidneys bearing 200 cultured islets was 7.88 +/- 1.6 mU (n = 13) versus 6.84 +/- 1.43 mU (n = 13) for kidneys bearing cryopreserved islets. Thus this technique for cryopreservation of isolated adult human islets enables a high recovery of endocrine tissue that survives after transplantation to nude rats, but some evidence of damage was apparent from insulin secretion studies and electron-microscopic studies.     

Vascular endothelial growth factor increases functional beta-cell mass by improvement of angiogenesis of isolated human and murine pancreatic islets

BACKGROUND: Blood flow is impaired in islet transplants, but there is conflicting evidence on improving the outcome by promoting vascularization. We previously reported that islet endothelial cells (EC) possess significant angiogenic capacity. METHODS: To further address this issue, we studied human islets in culture under hypoxic conditions. Moreover, we used a transgene mouse model with human vascular endothelial growth factor (VEGF) production in beta-cells under the control of the rat insulin promoter (RIP) to stimulate islet EC proliferation. RESULTS: Subsequent to a hypoxic stimulus, islets responded with specific expression patterns of VEGF and fibroblast growth factor; however, this was not sufficient to prevent the decay of islet EC. VEGF release of RIP-VEGF transgenic islets was controlled by glucose and resulted in the formation of sprouts. When transplanted to the kidney capsule of diabetic mice, RIP-VEGF islets significantly enhanced microvascular density and functional blood flow to the graft compared with controls. CONCLUSIONS: Optimized angiogenesis of islet transplants resulted in greater availability of insulin caused by beta-cell proliferation and a significantly higher percentage (90% versus 20%) of mice cured from diabetes.     

血管紧张素Ⅱ通过抑制AKT/蛋白激酶B磷酸化诱导肾小管上皮细胞凋亡

目的观察血管紧张素Ⅱ(AngⅡ)在诱导肾小管上皮细胞凋亡的信号传导是否有磷脂酰肌醇3激酶(PI3K)-AKT信号系统的参与,及该系统在诱导细胞凋亡中的作用。方法大鼠肾小管上皮细胞株NRK-52E分别与终浓度为0(对照组)、10-9mol/L、10-8mol/L、10-7mol/L、10-5mol/LAngⅡ共培养24h。用流式细胞仪检测细胞凋亡指数,用免疫组化方法检测增殖细胞核抗原(PCNA)的表达。Western印迹检测PI3K及磷酸化AKT和总AKT蛋白表达,AKT的磷酸化水平用473位丝氨酸磷酸化AKT(AKT-ser473)水平与总AKT水平的比值表示。结果随着AngⅡ浓度的增加,10-6mol/LAngⅡ组与对照组相比,凋亡指数显著增加[(22.7±1.41)%比(3.0±0.75)%,P<0.01]。而10-9mol/LAngⅡ组与对照组相比,PCNA指数显著增强[(47.54±2.6)%比(22.63±2.5)%,P<0.01]。与对照组相比,PI3K-p85蛋白的表达随AngⅡ浓度增加表现为先激活后抑制。AKT的磷酸化具有明显的AngⅡ浓度依赖性,随AngⅡ浓度的增加而逐渐受到抑制,并与细胞凋亡指数呈显著负相关(r=-0.90,P<0.01)。结论AngⅡ可以诱导肾小管上皮细胞凋亡并抑制细胞的增殖,可能部分是通过抑制PI3K-AKT信号传导途径实现的。     

Arachidonic acid metabolism and insulin secretion by isolated human pancreatic islets

Isolated human pancreatic islets converted [3H8]arachidonate to compounds with the high-performance liquid-chromatographic mobility of cyclooxygenase products, including prostaglandin E2 (PGE2), PGF2 alpha, and the lipoxygenase product 12-HETE. Human islet synthesis of PGE2, PGF2 alpha, and 12-HETE from endogenous arachidonate was demonstrated with stable isotope dilution-gas chromatographic-negative ion-chemical ionization-mass spectrometric analysis. Pharmacologic inhibition of arachidonate metabolism by both lipoxygenase and cyclooxygenase pathways with BW 755C strongly suppressed glucose-induced insulin secretion from perifused human islets, and the selective cyclooxygenase inhibitor indomethacin enhanced insulin secretion. These findings are similar to those reported for islets isolated from rats and suggest that arachidonate metabolites may modulate glucose-induced insulin secretion in humans.     

Water and cryoprotectant permeability characteristics of isolated human and canine pancreatic islets

Cryopreservation allows accumulation of the necessary islet transplantable mass as well as adequate time for tissue typing and infectious disease screening. Cryopreservation protocols may be optimized by modeling the osmotically induced volume excursions that occur during the addition and removal of cryoprotective agents (CPAs). To that end, three transport parameters were measured at 22 degrees C in canine and human islets isolated by collagenase digestion and euroficoll purification: (i) the apparent hydraulic conductivity (Lp), (ii) the permeability coefficient of the CPA (Ps), and (iii) the associated reflection coefficient (sigma). The parameters were determined by volumetric analysis of islets upon abrupt exposure to 1, 2, and 3 M dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), and propylene glycol (PG). The parameters were calculated using the Kedem-Katchalsky theory to describe islet volume excursion kinetics (assuming islets to be single equivalent osmotic units with the same volume and surface area of the actual islet) and a three-parameter curve fit was performed using the Marquardt-Levenberg method. It was determined that the permeability characteristics of pancreatic islets are species specific, and based upon the measured parameters, the highest Ps values for canine islets were observed following exposure to 2 M EG, and the highest Ps values for human islets were observed following exposure to 2 M PG. The permeability parameters were analyzed adjusting for islet radius using ANCOVA procedures to acquire least square means. For canine islets exposed to 2 M EG these values were determined to be 0.936 microm/min/atm, 2.47 microm/s, and 0.90 (for Lp, Ps, and phi, respectively) and for human islets exposed to 2 M PG the values were determined to be 1.56 microm/min/atm, 3.48 microm/s, and 0.85 (for Lp, Ps, and sigma, respectively). These parameters were used in a model to calculate osmotically induced islet volumetric response upon addition/dilution of the optimum CPAs, taking into consideration critical volume excursion limits at which irreversible damage occurs.     

Protein kinase C and calcium regulation of adenylyl cyclase in isolated rat pancreatic islets.

Rat islets express several isoforms of adenylyl cyclase (AC), and the regulation of AC activity in isolated islets by Ca(2+) and protein kinase C (PKC) was investigated. At basal 2.8 mmol/l glucose, the muscarinic receptor agonist carbamylcholine chloride (CCh) evoked a concentration-dependent increase in cAMP generation with a maximum increase at least 4.5-fold above control. In contrast, forskolin and glucagon-like peptide 1 fragment 7-36 amide increased cAMP accumulation 23-fold and almost 10-fold, respectively. Cholecystokinin 26-33 sulfated amide (CCK) also stimulated cAMP production by up to eightfold, as did the phorbol ester, phorbol 12,13-dibutyrate (PDBu). PDBu and CCh or CCK responses were not additive. The effects of phorbol ester, CCh, and CCK were inhibited by as much as 75% by the PKC inhibitors GF 109203X and Ro-32-0432 and after PKC downregulation. In the absence of extracellular Ca(2+), PDBu-, CCh-, and CCK-induced cAMP production was inhibited by approximately 50% in each case. Chelation of intracellular Ca(2+) with 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM) inhibited CCh- and CCK-stimulated cAMP generation by approximately 50% but did not inhibit the stimulatory effect of PDBu. Stringent Ca(2+) depletion by removal of extracellular Ca(2+) and inclusion of BAPTA/AM allowed for increased cAMP production in response to CCh and CCK; PKC inhibitors and PKC downregulation prevented this stimulation. Glucose stimulation also increased islet cAMP production, but PDBu did not potentiate the glucose response. The results suggest that Ca(2+) influx, Ca(2+) mobilization, and PKC activation play important roles in the modulation of AC activity in pancreatic islets.     

Molecular mechanisms of cell death of mycophenolic acid-treated primary isolated rat islets: implication of mitogen-activated protein kinase activation

Optimal immunosuppression after pancreas islet transplantation has not yet been established to achieve long-term graft survival. Mycophenolic acid (MPA) is widely used as an immunosuppressive drug after transplantation including among recipients of pancreas islet cells. Previously, we reported MPA-induced islet apoptosis in the HIT-T15 cell line. In this study, we confirmed the effects of MPA on cell death and its potential implications on the mitogen-activated protein kinase (MAPK) family expression levels in primary isolated rat islets. Lewis islets isolated by collagenase digestion were purified by the density gradient method. Cell death was analyzed by methylthiazoletetrazolium assay. Activation of MAPK kinase 4 (MKK4), c-jun N-terminal protein kinase (JNK), p38 MAPK, and caspase-3 cleavage was examined by Western blot analyses. MPA treatments (>25 μmol/L) increased cell death significantly at 24 hours and in a dose-dependent manner activated MKK4, JNK, and p38 MAPK at 20 hours. Caspase-3 cleavage was also increased by MPA treatment. These results suggested that MPA induced significant cell death among primary isolated rat islets by activation of MKK4, JNK, and p38 MAPK, as well as caspase-3 cleavage.     

Lessons from in vitro perifusion of pancreatic islets isolated from 80 human pancreases

We report the average insulin response to acute glucose measured by in vitro perifusion of pancreatic islets isolated from 80 consecutive human organs. Different perifusion parameters were considered [basal release, stimulation index (SI), time to peak, incremental area under the curve delta-AUC alpha)], and the correlation among them was determined. SI positively correlated with delta-AUC alpha (p < 0.001, r = 0.80) while negatively with time to peak (p < 0.05, r = -0.23). We also evaluated several variables of the isolation procedure that might affect responsiveness to glucose by human islets. Sex and age of pancreas donors, cold ischemia time, duration of the digestion, collagenase concentration, and lot characteristics (collagenase, trypsin, clostripain, and proteases activity), and final islet yield were considered. Multivariate regression analysis showed only an independent association between SI and the concentration of collagenase (p = 0.01).     

Hyperoxia improves the survival of intraportally transplanted syngeneic pancreatic islets

BACKGROUND: Hypoxia in the portal vein may compromise the survival of intraportally transplanted pancreatic islets. We therefore examined the effect of inspired oxygen on the outcome of islet transplantation. METHODS: Blood glucose concentrations, glucose tolerance, and the size and number of surviving islets were measured in diabetic rats housed for 48 hr under hyperoxic (100% O(2)), hypoxic (11% O(2)), or normoxic (21%O(2)) conditions after intraportal transplantation of 350, 500, 700, or 1,000 syngeneic islets. RESULTS: In normoxic diabetic rats, the smallest graft size to consistently restore normoglycemia was 1,000 islets. A graft size of 700 islets was effective in only three of nine animals, whereas 500 islets were ineffective in all eight animals undergoing transplantation. In contrast, in hyperoxically housed rats, graft sizes of 700 or 500 islets restored normoglycemia in eight of nine or five of eight animals, respectively. In those animals that became normoglycemic, the glucose tolerance of the hyperoxically treated rats receiving 700 islets was almost identical to that of normoxically housed animals receiving 1,000 islets. The average size of the islets 6 weeks after transplantation was the same in livers of hyperoxic and control rats. However, the total islet area and number of islets engrafted in hyperoxic rats was significantly increased when compared with livers from normoxic animals receiving the same graft size, so the area in hyperoxic rats receiving 700 islets was not significantly different from normoxic recipients of 1,000 islets. CONCLUSIONS: Hyperoxia posttransplantation increases the number of islets that survive the engraftment process and allows normalization of plasma glucose levels with a smaller number of transplanted islets.     

Isolation of viable human pancreatic islets

This article reports a method for the isolation of viable pancreatic islets from the human pancreas. Isolated islets were obtained from human pancreata of cadavers, patients undergoing surgical operations, and fetuses, using a freehand microdissection procedure. Viability was assessed by light microscopy of sections stained with aldehyde fuchsin and by measuring the insulin output of islets in response to a glucose stimulus in vitro using a perifusion system. Ten pieces of cadaver pancreas were studied. Islets were isolated from 6 specimens and in 5 of these were shown to respond to a glucose stimulus in vitro. Histologically the islets showed minimal damage with slight degranulation of the beta cells. Five pieces of pancreas removed at operation were studied as well. Islets were isolated in all cases, but only 2 showed a response to a glucose stimulus. Pancreata from a 26-week and 34-week human fetus were also studied. It was not possible to microdissect islets from either case, but small pieces of pancreas from the 26-week fetus were shown to respond to a glucose stimulus by producing a significant increase in insulin output.

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Résumé Cet article décrit une méthode pour isoler des ilÔts de Langérhans viables à partir de pancréas humain. Une technique de microdissection manuelle a permis d'isoler ces ilÔts à partir de pancréas humains obtenus chez des patients opérés, chez des donneurs cadavériques et chez des foetus. Leur viabilité a été établie par l'examen en microscopie optique de sections tissulaires préparés à la fuchsine aldehyde et par mesure de la sécrétion d'insuline en réponse à une stimulation au glucose au moyen d'un système de périfusion in vitro. Dix échantillons de pancréas cadavériques ont été étudiés. On a réussi à isoler des ilÔts chez six d'entre eux et cinq ont répondu au test de stimulation au glucose in vitro. à l'examen histologique, les ilÔts ne présentaient que peu de modification et une légère dégranulation des cellules bÊta. Cinq spécimens de pancréas obtenus chirurgiculement ont été étudiés. On a pu isoler des ilÔts dans tous les cas, mais seulement deux ont répondu à la stimulation au glucose. Finalement on a aussi étudié des échantillons pancréatiques obtenus chez deux foetus humains de 24 et 36 semaines respectivement. Chez ni l'un ni l'autre il n'a été possible d'isoler des ilÔts, mais de petites tranches de tissus pancréatiques préparés chez le foetus de 26 semaines et soumis au test de stimulation au glucose ont augmenté significativement leur sécrétion d'insuline.

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Part of the cost of this project was defrayed by a grant from the Wellcome Trust.     

Glutathione peroxidase protein expression and activity in human islets isolated for transplantation

BACKGROUND: Overexpression of antioxidant enzymes has been reported to protect rodent beta cells from oxidative stress. However, very little is known about protein expression and activity of antioxidant enzymes in human islets. METHOD/RESULTS: Human islet protein levels by Western analysis and enzymatic activity for the key antioxidant enzymes superoxide dismutases (SODs), catalase, and glutathione peroxidase-1 (GPx) were examined. Enzyme protein expression and activity were in the order SODs > catalase > GPx. Human islet GPx protein expression was significantly less than that found for catalase (p < 0.0001) and levels of GPx activity were virtually undetectable. As glucose and estrogens have been proposed to alter antioxidant enzyme levels, we examined islet data from male and female donors separately and under varying glucose concentrations. We found significantly less (p < 0.001) GPx protein expression in islets from females compared to males, but no significant regulation by glucose in either gender. CONCLUSIONS: Human islets have very low protein and activity levels for GPx, the essential enzyme for protection against excessive levels of intracellular lipid peroxides. GPx mimetics may be especially valuable in providing human islets with the broadest spectrum of protection against oxidative stress during isolation and transplantation.     

Central necrosis in isolated hypoxic human pancreatic islets: evidence for postisolation ischemia

A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.