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Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.
No full textHuman β-cell gene profiling is a powerful tool for understanding β-cell biology in normal and pathological conditions. The assessment is complicated when isolated islets are studied because of contamination by non-β-cells and the exposure to the trauma of isolation that causes changes in gene expression. These limitations can be overcome by dissecting the β-cells from the pancreatic tissue directly using the laser capture microdissection (LCM) technique. LCM allows the sampling of specific cell types from tissue sections. The technique requires morphological criteria or specific stains for targeted cells, and the protocols must preserve the condition of the sought-after macromolecules. We have developed a protocol of rapid tissue dehydration followed by identification of human β-cells by their intrinsic autofluorescence, which allows laser microdissection for gene-profiling studies
Towards better understanding of the contributions of overwork and glucotoxicity to the beta-cell inadequacy of type 2 diabetes
No full textType 2 diabetes (T2D) is characterized by reduction of beta-cell mass and dysfunctional insulin secretion. Understanding beta-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when beta-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, beta-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. beta-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains beta-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human beta-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how beta-cell phenotype evolves as T2D develops and progresses
Effects of Streptozocin Diabetes and Diabetes Treatment by Islet Transplantation on In Vivo Insulin Signaling in Rat Heart
No full textIslet transplantation restores altered insulin signaling in skeletal muscle and heart of streptozotocin-diabetic rats.
No full textIslet transplantation under the kidney capsule corrects the defects in glycogen metabolism in both liver and muscle of streptozocin diabetic rats
No full textInsulin signalling abnormalities in the heart of insulin-deficient diabetic rats: effects of islet transplantation.
No full textEffects of streptozocin diabetes and diabetes treatment by islet transplantation on in vivo insulin signaling in rat heart
No full textThe insulin signaling cascade was investigated in rat myocardium in vivo in the presence of streptozocin (STZ)-induced diabetes and after diabetes treatment by islet transplantation under the kidney capsule. The levels of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta -subunit, insulin receptor substrate (IRS)-2, and p52(Shc) were increased in diabetic compared with control heart, whereas tyrosine phosphorylation of IRS-1 was unchanged. The amount of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the level of PI 3-kinase activity associated with IRS-2 were also elevated in diabetes, whereas no changes in IRS-1-associated PI 3-kinase were observed. Insulin-induced phosphorylation of Akt on Thr-308 was increased fivefold in diabetic heart, whereas Akt phosphorylation on Ser-473 was normal. In contrast with Akt phosphorylation, insulin-induced phosphorylation of glycogen synthase kinase (GSK)-3, a major cellular substrate of Akt, was markedly reduced in diabetes. In islet-transplanted rats, the majority of the alterations in insulin-signaling proteins found in diabetic rats were normalized, but insulin stimulation of IRS-2 tyrosine phosphorylation and association with PI 3-kinase was blunted. In conclusion, in the diabetic heart, 1) IRS-1, IRS-2, and p52(Shc) are differently altered, 2) the levels of Akt phosphorylation on Ser-473 and Thr-308, respectively, are not coordinately regulated, and 3) the increased activity of proximal-signaling proteins (i.e., IRS-2 and PI 3-kinase) is not propagated distally to GSK-3. Islet transplantation under the kidney capsule is a potentially effective therapy to correct several diabetes-induced abnormalities of insulin signaling in cardiac muscle but does not restore the responsiveness of all signaling reactions to insulin
Islet trasnplantation restores normal levels of insulin receptor and substrate tyrosine phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle and myocardium of streptozocin diabetic rats
No full textEffects of Streptozocin Diabetes and Diabetes Treatment by Islet Transplantation on In Vivo Insulin Signaling in Rat Heart
No full textInsulin signaling abnormalities in skeletal and cardiac muscles of diabetic rats: correction by transplantation of pancreatic islets.
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