Isolation of Rat Pancreatic Islets by Ductal Injection of Collagenase1

Sutton, Robert; Peters, Michael J.; McShane, Paul; Gray, Derek W. R.; Morris, Peter J.

doi:10.1097/00007890-198612000-00022

Cited by 247 publications

(140 citation statements)

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“…Cell culture Rat insulinoma cell line INS-1 cells were cultured and treated with 600 μmol/l stearic or other fatty acids for 24 h. Mouse pancreatic islets were isolated as described previously [23]. See ESM Methods.…”

Section: Methodsmentioning

confidence: 99%

Elevated circulating stearic acid leads to a major lipotoxic effect on mouse pancreatic beta cells in hyperlipidaemia via a miR-34a-5p-mediated PERK/p53-dependent pathway

Hao

et al. 2016

Diabetologia

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Aims/hypothesis Serum stearic acid (C18:0) is elevated in individuals with hyperlipidaemia and type 2 diabetes. However, the lipotoxicity induced by increased stearic acid in beta cells has not been well described. This study aimed to examine the adverse effects of stearic acid on beta cells and the potential mechanisms through which these are mediated. Methods Three groups of C57BL/6 mice were fed a normal diet or a high-stearic-acid/high-palmitic-acid diet for 24 weeks, respectively. The microRNA (miR) profiles of islets were determined by microarray screening. Islet injury was detected with co-staining using the TUNEL assay and insulin labelling. A lentiviral vector expressing anti-miRNA-34a-5p oligonucleotide (AMO-34a-5p) was injected into mice via an intraductal pancreatic route. Results In both mouse islets and cultured rat insulinoma INS-1 cells, stearic acid exhibited a stronger lipotoxic role than other fatty acids, owing to repression of B cell CLL/ lymphoma 2 (BCL-2) and BCL-2-like 2 (BCL-W) by stearic acid stimulation of miR-34a-5p. The stearic-acid-induced lipotoxicity and reduction in insulin secretion were alleviated by AMO-34a-5p. Further investigations in INS-1 cells revealed that p53 was involved in stearic-acid-induced elevation of miR-34a-5p, owing in part to activation of protein kinaselike endoplasmic reticulum kinase (PERK). Conversely, silencing PERK alleviated stearic-acid-induced p53, miR-34a-5p and lipotoxicity. Conclusions/interpretation These findings provide new insight for understanding the molecular mechanisms underlying not only the deleterious impact of stearic-acid-induced lipotoxicity but also apoptosis in beta cells and progression to type 2 diabetes.

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Section: Methodsmentioning

confidence: 99%

Elevated circulating stearic acid leads to a major lipotoxic effect on mouse pancreatic beta cells in hyperlipidaemia via a miR-34a-5p-mediated PERK/p53-dependent pathway

Hao

et al. 2016

Diabetologia

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“…All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee, Department of Territory, State of Geneva. Mouse islets were isolated and purified using techniques previously described [17,24]. Viability, as assessed by propidium iodide/fluorescein diacetate staining, was always higher than 80%.…”

Section: Methodsmentioning

confidence: 99%

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

Wojtusciszyn

Armanet²,

Morel³

et al. 2008

Diabetologia

127

102

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Aims/hypothesis We assessed the heterogeneity of insulin secretion from human isolated beta cells and its regulation by cell-to-cell contacts. Methods Insulin secretion from single and paired cells was assessed by a reverse haemolytic plaque assay. The percentage of plaque-forming cells, the mean plaque area and the total plaque development were evaluated after 1 h of stimulation with different secretagogues. Results Not all beta cells were surrounded by a haemolytic plaque under all conditions tested. A small fraction of the beta cell population (20%) secreted more than 90% and 70% of total insulin at 2.2 and 22.2 mmol/l glucose, respectively. Plaque-forming cells, mean plaque area and total plaque development were increased at 12.2 and 22.2 compared with 2.2 mmol/l glucose. Insulin secretion of single beta cells was similar at 12.2 and 22.2 mmol/l glucose. Insulin secretion of beta cell pairs was increased compared with that of single beta cells and was higher at 22.2 than at 12.2 mmol/l glucose. Insulin secretion of beta cells in contact with alpha cells was also increased compared with single beta cells, but was similar at 22.2 compared with 12.2 mmol/l glucose. Delta and other non-beta cells did not increase insulin secretion of contacting beta cells compared with that of single beta cells. Differences in insulin secretion between 22.2 and 12.2 mmol/l glucose were observed in murine but not in human islets. Conclusions/interpretation Human beta cells are highly heterogeneous in terms of insulin secretion so that a small fraction of beta cells contributes to the majority of insulin secreted. Homologous and heterologous intercellular contacts have a significant impact on insulin secretion and this could be related to the particular architecture of human islets.

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“…Islets were isolated after digestion of the pancreas by intraductal injection of 1 mg/ml collagenase P (Roche Molecular Biochemicals) dissolved in Krebs-Ringer bicarbonate buffer (KRBB) solution as described elsewhere (15,16). Collected islets were used directly or dispersed mechanically into single cells in a Ca 2ϩ -free KRBB solution for electrophysiological measurements.…”

Section: and Sur1mentioning

confidence: 99%

Sur1 Knockout Mice

Seghers¹,

Nakazaki²,

DeMayo³

et al. 2000

Journal of Biological Chemistry

347

124

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Sur1 knockout mouse ␤-cells lack K ATP channels and show spontaneous Ca 2؉ action potentials equivalent to those seen in patients with persistent hyperinsulinemic hypoglycemia of infancy, but the mice are normoglycemic unless stressed. Sur1 -/-islets lack first phase insulin secretion and exhibit an attenuated glucose-stimulated second phase secretion. Loss of the first phase leads to mild glucose intolerance, whereas reduced insulin output is consistent with observed neonatal hyperglycemia. Loss of K ATP channels impairs the rate of return to a basal secretory level after a fall in glucose concentration. This leads to increased hypoglycemia upon fasting and contributes to a very early, transient neonatal hypoglycemia. Whereas persistent hyperinsulinemic hypoglycemia of infancy underscores the importance of the K ATP -dependent ionic pathway in control of insulin release, the Sur1 -/-animals provide a novel model for study of K ATP -independent pathways that regulate insulin secretion.ATP-sensitive potassium channels (K ATP channels) 1 are a unique combination of a K ϩ inward rectifier (either K IR 6.1 or K IR 6.2) and a sulfonylurea receptor (SUR1 or SUR2) transport ATPase superfamily members (1-3). These channels respond to changes in ATP/ADP and can couple metabolism to membrane electrical activity. SUR1 and K IR 6.2 comprise the K ATP channels in pancreatic ␤-cells that regulate the ionic pathway mediating glucose-stimulated insulin secretion by setting the resting membrane potential below the activation threshold for voltage-gated Ca 2ϩ channels (4). Mutations in human Sur1 or K IR 6.2 cause a recessive form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) characterized by oversecretion of insulin despite severe hypoglycemia (1, 5). Surprisingly, two recent studies involving disruption of K ATP channels in mice produced a quite different picture. Targeted overexpression of a dominant-negative K IR 6.2 subunit, K IR 6.2 G132S , in ␤-cells reduced channel activity producing animals that were hypoglycemic at birth but became increasingly hyperglycemic secondary to ␤-cell death (6).K IR 6.2 null mice, K IR 6.2 -/-, on the other hand, completely lack ␤-cell K ATP channels but exhibit a less severe phenotype (7). The K IR 6.2 -/-animals have nearly normal blood glucose levels, showing mild glucose intolerance when challenged with glucose. These animals are reported to release a small amount of insulin in response to glucose, whereas isolated, perifused islets show a small first phase of glucose-stimulated insulin secretion and no second phase. The normal blood glucose levels have been attributed to insulin hypersensitivity secondary to the loss of SUR2A/K IR 6.2 K ATP channels in skeletal muscle.SUR1 null mice, Sur1 -/-, unlike their K IR 6.2 -/-counterparts, are not insulin-hypersensitive. Isolated Sur1 -/-islets exhibit a pattern of glucose-stimulated insulin release consistent with regulation by an underlying K ATP -independent pathway (or pathways), the nature of which is unknown (8 -12). The Sur...

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Isolation of Rat Pancreatic Islets by Ductal Injection of Collagenase1

Cited by 247 publications

References 0 publications

Elevated circulating stearic acid leads to a major lipotoxic effect on mouse pancreatic beta cells in hyperlipidaemia via a miR-34a-5p-mediated PERK/p53-dependent pathway

Elevated circulating stearic acid leads to a major lipotoxic effect on mouse pancreatic beta cells in hyperlipidaemia via a miR-34a-5p-mediated PERK/p53-dependent pathway

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

Sur1 Knockout Mice

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