Figure 3. The recruitment of proinsulin mRNA on to the membranes at high glucose plays a significant role in glucose-stimulated proinsulin synthesis.

MIN6 cells were preincubated in KRB containing 0.5 mM glucose for 1 h followed by incubation in KRB containing 0.5 or 20 mM glucose for a further period of 1 h. Membranes were isolated by consecutive centrifugation as described in Figure 1. (a) Resuspended membranes were translated in situ in the presence of [³⁵S]methionine. After translation, the membranes were pelleted at 13000 g for 10 min. Proinsulin (PI) was resolved by SDS/PAGE. (b) Poly(A)⁺ selected mRNA was isolated from the membranes and translated in vitro in the presence of [³⁵S]methionine. Proinsulin (PI) was immunoprecipitated and resolved by SDS/PAGE. (c) MIN6 cells were preincubated in KRB containing 0.5 mM glucose for 1 h followed by incubation in KRB containing 0.5 or 20 mM glucose in the presence of [³⁵S]methionine for a further period of 1 h. The cells were lysed and proinsulin (PI) resolved by SDS/PAGE. In all cases, proteins were visualized by autoradiography. In (a, c) the results are representative of three separate experiments and in (b) the results are representative of two separate experiments.