Figure 4.

Leptin enhances long-lived protein degradation rate in adipocytes. (a) Time-dependent release of radioactive valine from epididymal adipocytes pre-pulsed with L-[¹⁴C(U)]-Valine, as described in Methods. Cells were either treated with EBSS+0.1% BSA to stimulate autophagy (= positive control), control media (con), media containing leptin (100 ng/ml) only, or media containing leptin + SMLA (1 μg/ml) with or without Baf.A. The radioactivity was measured in both the supernatant fraction and cell pellets and % of [¹⁴C]-valine release was calculated as described in Methods. (b) Long-lived protein degradation rate calculated as % of L-[¹⁴C(U)]-Valine release per 1 h, with or without Baf.A. (0.1 μM). Results are mean ± SEM of 3 independent experiments, each performed in triplicates. Different letters denote significant differences between treatments in the absence of Baf.A, p < 0.05. (c) Net autophagy-mediated L-[¹⁴C(U)]-Valine release (∆Protein degradation rate) as calculated from b. As in b, different letters denote significant differences between treatments (-Baf.A-(+Baf.A), p < 0.05.