FIG. 3.

Rald regulates Atgl protein levels in Aldh1a1^−/− male VF ex vivo and in differentiated 3T3-L1 adipocytes in vitro. A: Atgl protein levels in VF explants isolated from Aldh1a1^−/− males (n = 3) were measured by Western blot. Three VF pads dissected from each animal (equal by weight 76 mg) were immediately stimulated with vehicle, RA, and Rald (all 10 nmol/L) for 2 h. Atgl levels were normalized to housekeeping proteins β-actin or glyceraldehyde-3-phosphate dehydrogenase. Data for each set (inset) were normalized to vehicle control (100%) for VF from each animal. *Difference between control (Veh) and Rald-explants (P < 0.05) (all mean ± SE; n = 3); #difference between Rald- and RA-stimulated explants (P < 0.05). Visc, visceral. B: Atgl protein levels were analyzed by Western blot in VF from C57BL/6J (WT) ovariectomized and sham-operated female mice (all mean ± SE; n = 4). Inset shows estrogen (E2) levels in plasma using ELISA. C: Atgl mRNA expression levels were measured by TaqMan in the same VF. Mann-Whitney U test analysis showed no significance between groups. D: Atgl levels in differentiated (8 days) 3T3-L1 fibroblasts measured by Western blot. Cells were stimulated for 45 min with retinol (Rol), RA, and Rald (all retinoids 100 nmol/L) in 100 nmol/L insulin/20% FBS medium. Atgl levels were normalized to tubulin. Data are shown as a percent of vehicle control. Inset at top shows a representative example of a Western blot. *Difference between control (Veh) and Rald-stimulated cells; #difference between Rald- and RA-stimulated cells, both P < 0.05 (mean ± SE; n = 3). E: Atgl levels in differentiated (8 days) 3T3-L1 fibroblasts stimulated with cycloheximide (20 μg/mL) for 6 or 30 h. The cycloheximide-treated adipocytes were stimulated with vehicle and Rald (10 nmol/L) for an additional 45 min. Atgl and β-actin levels were measured by Western blot. Prolonged (30 h) cycloheximide treatment inhibited protein translation and abolished the Rald-dependent increase in Atgl protein levels.