Figure 8.

Biosynthesis of proinsulin and insulin in WT control and Ins2-proinsulin-R(B22)E heterozygous (Het) and homozygous (Hom) males with progression of diabetes (age 4–8 weeks). The random blood glucose of each animal at the time of euthanasia is indicated. Isolated islets were pulse labeled with ³⁵S-amino acids for 30 min and then chased for 2 h, as indicated. Cells (C) and chase media (M) were either combined (so that no protein was lost) or analyzed separately. Samples (normalized to trichloroacetic acid–precipitable counts in the cell lysates) were immunoprecipitated with anti-insulin followed by Tris-tricine-urea-SDS-PAGE under nonreducing or reducing conditions followed by fluorography. A line is drawn separating the nonreduced and reduced samples, but these images show the complete gels, and no lanes have been excised. A–C: Three independent experiments with the genotypes shown (ages 4, 5, and 8 weeks, respectively). D: Quantitative recovery of newly synthesized insulin derived from pulse-labeled proinsulin, as derived from the preceding phosphorimages; genotype and random blood glucose are indicated. Proinsulin bands (at chase time 0) were quantitated from reducing gels; newly synthesized insulin derived from the pulse-labeled samples were quantified from nonreducing gels. (Insulin is a two-chain protein that “falls apart” under reducing conditions; thus, nonreducing gels are preferable for this analysis.) ox, oxidized; R, reduced.