Figure 1.

Insulin stimulates ACRP30 secretion from 3T3-L1 adipocytes, but does not stimulate α3 (VI) collagen secretion. Three 10-cm plates of 3T3-L1 adipocytes were serum-starved and total cellular protein was labeled with a 15-min pulse of ³⁵S-labeled cysteine and methionine. Cells were washed and then chased in serum-free media containing cycloheximide to inhibit further protein synthesis. Additionally during the chase period, cells were maintained in the presence or absence of 160 nM insulin or 200 μM A23187. The media were collected and replaced at 30-min intervals, and the appearance of labeled ACRP30 or α3 (VI) collagen in the media was assessed by sequential immunoprecipitations. The immunoprecipitates were analyzed SDS-PAGE, and bands were detected and quantified using a PhosphorImager. A shows the bands corresponding to ACRP30 and α3 (VI) collagen for each condition. In B, these bands were quantified and the cumulative secretion into the media is plotted for each protein. The total amount of protein secreted by unstimulated cells is taken as 100% and all other data are plotted relative to this amount.