An Improved Method for Isolation of Mouse Pancreatic Islets

Gotoh, Mitsukazu; Maki, Takashi; Kiyoizumi, Takashi; Satoh, Susumu; Monaco, Anthony P.

doi:10.1097/00007890-198510000-00018

Cited by 537 publications

(299 citation statements)

References 0 publications

Supporting

Mentioning

297

Contrasting

Unclassified

Order By: Relevance

“…Immunostaining of pancreatic GPX1 protein (n =3 mice×three slides per genotype) was conducted using a rabbit polyclonal antibody against bovine GPX1 (Lab Frontier, Seoul, Korea). Islets were isolated from mice using a standard procedure [39] with minor modifications. Total GPX1 activity was assayed in homogenised islets isolated from the OE and WT mice (n=3 per genotype) [10].…”

Section: Methodsmentioning

confidence: 99%

Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice

et al. 2008

View full text Add to dashboard Cite

Aims/hypothesis We previously observed hyperglycaemia, hyperinsulinaemia, insulin resistance and obesity in Gpx1-overexpressing mice (OE). Here we determined whether these phenotypes were eliminated by diet restriction, subsequently testing whether hyperinsulinaemia was a primary effect of Gpx1 overexpression and caused by dysregulation of pancreatic duodenal homeobox 1 (PDX1) and uncoupling protein-2 (UCP2) in islets. Methods First, 24 male OE and wild-type (WT) mice (2 months old) were given 3 g (diet-restricted) or 5 g (fullfed) feed per day for 4 months to compare their glucose metabolism. Thereafter, several mechanistic experiments were conducted with pancreas and islets of the two genotypes (2 or 6 months old) to assay for beta cell mass, reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δ= m ) and expression profiles of regulatory proteins. A functional assay of islets was also performed. Results Diet restriction eliminated obesity but not hyperinsulinaemia in OE mice. These mice had greater pancreatic beta cell mass (more than twofold) and pancreatic insulin content (40%) than the WT, along with an enhanced Δ= m and glucose-stimulated insulin secretion in islets. With diminished ROS production, the OE islets displayed hyperacetylation of H3 and H4 histone in the Pdx1 promoter, elevated PDX1 and decreased UCP2. Conclusions/interpretation Overproduction of the major antioxidant enzyme, glutathione peroxidase 1, caused seemingly beneficial changes in pancreatic PDX1 and UCP2, but eventually led to chronic hyperinsulinaemia by dysregulating islet insulin production and secretion.

show abstract

Section: Methodsmentioning

confidence: 99%

Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Briefly, islets were isolated by digesting pancreases with collagenase P and purified on a discontinuous Ficoll density gradient as described by Gotoh et al [32]. Islets were hand-picked and transferred to DMEM supplemented with 10% FCS, 11 mmol/l glucose, 100 units/ml penicillin and 100 μg/ml streptomycin for culture.…”

Section: Methodsmentioning

confidence: 99%

Vascular endothelial growth factor as a survival factor for human islets: effect of immunosuppressive drugs

et al. 2007

View full text Add to dashboard Cite

Aims/hypothesis Rapamycin, part of the immunosuppressive regimen of the Edmonton protocol, has been shown to inhibit vascular endothelial growth factor (VEGF) production and VEGF-mediated survival signalling in tumour cell lines. This study investigates the survival-promoting activities of VEGF in human islets and the effects of rapamycin on islet viability.Materials and methods Levels of VEGF and its receptors in isolated human islets and whole pancreas was determined by western blotting and immunostaining. Islet viability following VEGF or immunosuppressive drug treatment was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Islet VEGF release was measured by ELISA. Mouse islets infected with an adenovirus expressing the gene for VEGF were transplanted syngeneically into streptozotocin-induced diabetic mice, with blood glucose levels measured three times per week. Results Isolated human islets produced multiple isoforms of VEGF and VEGF receptors 1, 2 and 3 and the coreceptor neuropilin 1. Exogenous VEGF (10 ng/ml) prevented human islet death induced by serum starvation, which suggests that VEGF can act as a survival factor for human islets. Transplantation of mouse islets infected with a VEGF-expressing adenovirus in a syngeneic model, improved glycaemic control at day 1 post-transplantation (p< 0.05). Rapamycin at 10 and 100 ng/ml significantly reduced islet VEGF release (by 37±4% and 43±6%, respectively; p<0.05) and at 100 ng/ml reduced islet viability (by 36±9%) and insulin release (by 47±7%, all vs vehicle-treated controls; p<0.05). Tacrolimus had no effect on islet VEGF release or viability. Conclusions/interpretation Our data suggest that rapamycin may have deleterious effects on islet survival posttransplantation, both through a direct effect on islet viability and indirectly through blockade of VEGF-mediated revascularisation.

show abstract

“…Pancreatic islets of Langerhans were isolated from male Wistar rats (200 -230 g) by perfusion of the pancreatic duct and in situ collagenase digestion [34]. Islets were subsequently purified by two Histopaque (Sigma) density gradients and hand picking, as described in [35]. Immediately after isolation, islets were cultured overnight in DMEM supplemented with 30 % (v\v) heat-inactivated foetal calf serum, 100 i.u.…”

Section: Min6 Cell Culture and Islet Isolationmentioning

confidence: 99%

Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression

Xavier

Leclerc

Váradi

et al. 2003

Biochemical Journal

245

238

View full text Add to dashboard Cite

AMP-activated protein kinase (AMPK) has recently been implicated in the control of preproinsulin gene expression in pancreatic islet β-cells [da Silva Xavier, Leclerc, Salt, Doiron, Hardie, Kahn and Rutter (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4023–4028]. Using pharmacological and molecular strategies to regulate AMPK activity in rat islets and clonal MIN6 β-cells, we show here that the effects of AMPK are exerted largely upstream of insulin release. Thus forced increases in AMPK activity achieved pharmacologically with 5-amino-4-imidazolecarboxamide riboside (AICAR), or by adenoviral overexpression of a truncated, constitutively active form of the enzyme (AMPKα1.T172D), blocked glucose-stimulated insulin secretion. In MIN6 cells, activation of AMPK suppressed glucose metabolism, as assessed by changes in total, cytosolic or mitochondrial [ATP] and NAD(P)H, and reduced increases in intracellular [Ca2+] caused by either glucose or tolbutamide. By contrast, inactivation of AMPK by expression of a dominant-negative form of the enzyme mutated in the catalytic site (AMPKα1.D157A) did not affect glucose-stimulated increases in [ATP], NAD(P)H or intracellular [Ca2+], but led to the unregulated release of insulin. These results indicate that inhibition of AMPK by glucose is essential for the activation of insulin secretion by the sugar, and may contribute to the transcriptional stimulation of the preproinsulin gene. Modulation of AMPK activity in the β-cell may thus represent a novel therapeutic strategy for the treatment of type 2 diabetes mellitus.

show abstract

An Improved Method for Isolation of Mouse Pancreatic Islets

Cited by 537 publications

References 0 publications

Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice

Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice

Vascular endothelial growth factor as a survival factor for human islets: effect of immunosuppressive drugs

Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression

Contact Info

Product

Resources

About