Identification of glucose response proteins in two biological models of beta-cell adaptation to chronic high glucose exposure.
High resolution 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with computerized analysis of gel images was used to search for proteins whose biosynthesis was induced or repressed in pancreatic islet cells chronically exposed to high glucose in an in situ and a tissue culture model of islet cell adaptation to excessive fuel load. The in situ model involved a 4-day intravenous infusion of either 50% glucose or 0.45% saline solution, followed by islet isolation, [35S]methionine labeling at 3 and 18 mM glucose for both groups, and protein analysis by 2-dimensional SDS-PAGE. The tissue culture model involved a 7-day culture of isolated rat islets in RPMI 1640 with 10% fetal calf serum containing either 3 or 30 mM glucose, followed by radiolabeling and 2-dimensional PAGE of proteins as in the in situ model. A small fraction of about 1.5% of the approximately 2000 identifiable proteins can be characterized as adaptive proteins. Of these altogether 58 proteins in the two models, 5 proteins were demonstrable in both models and two of these (proteins 1526 and 7622) are particularly noteworthy. Protein 1526 (Mr 57,000; pI 5.09) showed the same response pattern in both models and its expression was most enhanced when islets from chronically glucose-infused animals or those cultured for 7 days at 30 mM were radiolabeled at 18 mM glucose. Protein 7622 (Mr 68,000; pI 6.50) (also known as GSP-65; Collins, H.W., Buettger, C., and Matschinsky, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5494-5498) showed a different labeling pattern in the two models: stimulation of [35S]methionine incorporation by 18 mM glucose both in control and experimental islets from the infusion study, but lack of such stimulation of radiolabeling in islets cultured for 7 days at 30 mM glucose in contrast to islets cultured at 3 mM. The experimental strategy and the methodology are evaluated and the significance of the results is discussed. Potentials of the approach and plans for future experiments are considered.
Collins
H
H
Diabetes Research Center, University of Pennsylvania School of Medicine, Philadelphia 19104-6015.
Najafi
H
H
Buettger
C
C
Rombeau
J
J
Settle
R G
RG
Matschinsky
F M
FM
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D005947:Glucose / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000096:biosynthesis / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008954:Models, Biological; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D011506:Proteins; D005947:Glucose