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Fig. 1. Effects of chronic high glucose treatment on insulin secretion and gene expression in INS-1E cell. (A) INS-1E cells in 24-well dishes were cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours. After 5 hours equilibration in 2.5 mM glucose medium, cells were washed twice with KRBH. Insulin release from INS-1E cells stimulated with 20 mM glucose, 20 mM leucine and 20 mM KCl in KRBH buffer containing 2.5 mM (referred to Basal) was determined by radio-immunoassay and expressed as a percentage of cellular insulin content. The equilibration period is necessary to see the glucose-responsiveness of insulin release in INS-1 cells. Cellular insulin content was decreased by 32.4±6.7% after 48 hours treatment with 30 mM glucose. Data represent mean ± s.e.m. of six independent experiments. **P<0.001. (B,C) The gene expression patterns in INS-1E cells cultured in either standard (11.2 mM) or 30 mM glucose medium for 48 hours were quantified by northern blotting. Total RNA samples (20 µg/lane) were analysed by hybridizing with the indicated cDNA probes. Four independent experiments are shown side by side to demonstrate the consistency of the results.
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