Fig. 7. Expression of Hub increases C-peptide degradation (relative to insulin) at later times of chase and in the steady state. Cells were infected with adenovirus and sorted as described in the legend to Fig. 3. (A) Pulse-chase. The sorted cells were labelled (10 minutes [³H]leucine) and chased (150 minutes basal followed by 60 minutes stimulated). The two chase media were analyzed by HPLC. The data (mean±s.e.m., n=4) are for the ratio of labelled insulin to total (intact plus truncated) labelled C-peptide (corrected for the difference in the number of leucines in each). ^*P<0.02 for Hub vs control for products released during the 60 minute chase with secretagogues (`stimulated'). (B) Steady-state. The sorted cells were incubated for 150 minutes under basal conditions and then for a further 60 minutes with secretagogues. In order to estimate the ratio of (unlabelled) insulin:C-peptide, the 60 minute stimulated medium was analysed by HPLC and the peaks monitored by an online spectrophotometer at 213 nm. Peak areas for C-peptide I and insulin I were measured. The insulin:C-peptide ratio for cells expressing Hub was normalized to that for control cells (for which the measured value was 2.45±0.27). Data are presented as mean±s.e.m., n=5 from four independent experiments. ^*P<0.005.

Return to article