title: A modified method of islet isolation preserves the ability of pancreatic islets to increase protein O-glycosylation in response to glucose and streptozotocin.
abstract: An important link has recently been shown in vivo between beta-cell O-linked protein glycosylation and beta-cell apoptosis, with hyperglycemia having been demonstrated to reversibly increase beta-cell O-linked protein glycosylation by providing substrate for the glucosamine pathway. In contrast, the same study showed that the administration of streptozotocin to rats prior to the induction of hyperglycemia results in irreversible increases in O-glycosylation and subsequent beta-cell apoptosis. In light of these data, we investigated beta-cell O-glycosylation in vitro by exposing isolated rat islets to high glucose, glucosamine, or streptozotocin and analyzing the pattern of O-glycosylated proteins present. All three compounds acutely increased O-glycosylation of a predominate 135-kDa protein (p135); however, their ability to stimulate p135 O-glycosylation was only consistently observed when islets were isolated in the presence of high glucose and 1 mM L-glutamine. Islets isolated in low glucose and no added L-glutamine demonstrated no consistent increase in p135 O-glycosylation in response to glucose, glucosamine, or streptozotocin. These data suggest that during islet isolation, beta-cell enzymes responsible for regulating p135 O-glycosylation may be adversely affected by the absence of high glucose and glutamine, which together are necessary for O-linked N-acetylglucosamine synthesis. We propose that the combination of high glucose and glutamine during islet isolation generates UDP-N-acetylglucosamine and O-linked N-acetylglucosamine, thus providing substrate protection for these enzymes and preserving the ability of isolated islets to O-glycosylate p135.
authors.affiliation: Department of Pathology, University of Alabama at Birmingham School of Medicine, 35233-7331, USA. rkonrad@path.uab.edu
authors.forename: R J
authors.initials: RJ
authors.lastname: Konrad
authors.forename: K
authors.initials: K
authors.lastname: Liu
authors.forename: J E
authors.initials: JE
authors.lastname: Kudlow
mesh_terms: D000818:Animals; D005944:Glucosamine / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D005973:Glutamine / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013311:Streptozocin / Q000633:toxicity*; D014021:Tissue Preservation / Q000379:methods*
chemical_list: D011506:Proteins; D005973:Glutamine; D013311:Streptozocin; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D005947:Glucose; D005944:Glucosamine