Islet isolation assessment in man and large animals

Ricordi, Camillo; Gray, Derek W. R.; Hering, Bernhard J.; Kaufman, Dixon B.; Warnock, Garth L.; Kneteman, Norman M.; Lake, S. P.; London, N. J. M.; Socci, C.; Alejandro, Rodolfo; Zeng, Yijun; Scharp, David W.; Viviani, Giorgio Luciano; Falqui, Luca; Tzakis, Andreas G.; Bretzel, Reinhard G.; Federlin, K.; Pozza, G.; James, R. F. L.; Rajotte, Ray V.; Carlo, V. Di; Morris, Peter J.; Sutherland, David E.R.; Starzl, Thomas E.; Mintz, Daniel H.; Lacy, Paul E.

doi:10.1007/bf02581331

Cited by 559 publications

(462 citation statements)

References 36 publications

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“…Freshly isolated islets were treated with either TAT peptide alone or with L-JNKI (10 μmol/l; BioSynthesis, Lewisville, TX, USA). After overnight culture, islet counts were obtained and expressed as islet equivalents (IEQ) after diphenylthiocarbazone staining [21,22]. Data were expressed as percentage of islet loss as compared with 100% placed in culture at day 0.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

et al. 2007

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Aims/hypothesis Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cellpermeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells. Methods The effect of L-JNKI (10 μmol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied. Results In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection. Conclusions/interpretation In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.

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Section: Methodsmentioning

confidence: 99%

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

et al. 2007

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“…Islets were stained with diphenyl-thiocarbazone, counted at the microscope and the number expressed as absolute number (NA) and equivalent number (NE; the absolute number corrected for coefficients based on islet volume, [11]). According to their morphology, islets were divided into two categories: (1) roundshaped, when they maintained their morphology and a large portion of the peripheral capsule; (2) fragmented, when they showed architectural disorganisation with irregular borders and free chains of cells protruding from the periphery.…”

Section: Subjects Materials and Methodsmentioning

confidence: 99%

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

et al. 2005

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Aims/hypothesis: Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation. Methods: Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome. Results: By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p= 0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p= 0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p= 0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p= 0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p= 0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071). Conclusions/interpretation: These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

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“…Two media changes were carried during that period. Islet volume and purity were determined by microscopical sizing on a grid after staining with diphenylthiocarbazone [16] and viability was assessed by membrane integrity testing (trypan blue exclusion). Islets were shipped to the department of Molecular Medicine at Karolinska Institutet, Stockholm, Sweden, where a cell suspension was prepared [17].…”

Section: Methodsmentioning

confidence: 99%

Long-Chain CoA esters activate human pancreatic beta-cell K ATP channels: potential role in Type 2 diabetes

et al. 2004

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Aims/hypothesis. The ATP-regulated potassium (K ATP ) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the K ATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent K ATP channel activators in rodents, as studied in inside-out patches. Methods. In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. Results. Human K ATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg 2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC 50 of 0.7±0.1 µmol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell K ATP channel currents increased approximately sixfold following addition of 1 µmol/l LC-CoA ester. The presence of 1 µmol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5±0.2 nS to 2.5±1.3 nS. Conclusion/interpretation. Taken together, these results show that LC-CoA esters are potent activators of the K ATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate K ATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions. [Diabetologia (2004) essential function has been included in the model of glucose-induced insulin secretion. In this model, increases in blood glucose levels lead to metabolism of glucose which generates an increase in the ATP/ADP ratio. The consensus view is that increases in the ATP/ADP ratio promote closure of the beta-cell K ATP channel, thereby depolarizing the cell membrane resulting in opening of voltage-dependent Ca 2+ channels. The subsequent rise in cytoplasmic free Ca 2+ concentration triggers a series of events leading to exocytosis of insulin. Hence, the K ATP channel provides a critical link between glucose metabolism, electrical activity and insulin secretion in the pancreatic beta cell.The most important molecular regulators of K ATP channel activity are believed to be adenine nucleo-ATP-sensitive potassium (K ATP ) channels play a key role in the coupling between cellular metabolism and electrical activity in a wide range of tissues. Since the discovery of these channels in the beta cell [1], their

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Islet isolation assessment in man and large animals

Cited by 559 publications

References 36 publications

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

Long-Chain CoA esters activate human pancreatic beta-cell K ATP channels: potential role in Type 2 diabetes

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