Characterization of insulin glycation in insulin-secreting cells maintained in tissue culture.

Characteristics of cellular insulin glycation were examined in the pancreatic B-cell line, BRIN-BD11. The extent of insulin glycation increased stepwise during 72 h of culture at 5.6-33.3 mmol/l glucose, attaining levels up to 27%. Glycation of insulin at 33.3 mmol/l glucose was rapid, reaching maximal values within 2 h, and not readily reversible during 2 to 24 h of subsequent exposure to 5.6 mmol/l glucose. Glycated insulin was readily secreted by BRIN-BD11 cells upon active stimulation with glucose and other secretagogues. Cellular insulin glycation was decreased by 66-80% by inhibitors of protein glycation, vitamin C, aminoguanidine or acetylsalicylic acid. Modulation of insulin-secretory activity of BRIN-BD11 cells by co-culture at high glucose with diazoxide. L-alanine or glibenclamide indicated that long-term stimulation of secretion was associated with a decrease in the extent of insulin glycation. Glycation of insulin in vitro was substantially less extensive than in BRIN-BD11 cells, although glucose-6-phosphate and glyceraldehyde-3-phosphate were 1.4- to 2.0-fold more reactive than glucose per se. These observations indicate that insulin is readily glycated and secreted from insulin-secreting cells under hyperglycaemic conditions in culture.