Acute insulin stimulation of ATX secretion is PI3Kinase-dependent and mTOR-independent. 3T3-L1 adipocytes were incubated with media containing 25 mM glucose in the presence or absence of 0 or 100 nM insulin for 6 hours. Where indicated, adipocytes were additionally treated with either 1 µM wortmannin (W), 100 nM rapamycin (R), 10 µg/mL cycloheximide (CHX), or a combination of 5 µg/mL brefeldin A and 5 µM monensin (B/M). (a, b) Cellular ATX (ATX-C) protein expression, (c, d) secreted ATX (ATX-S) protein expression, and (e) activity. Statistical analysis was performed using one-way analysis of variance followed by a Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs untreated controls; ^##P < 0.01; ^###P < 0.001; ^####P < 0.0001 for insulin plus chemical inhibitors vs insulin minus chemical inhibitors; n = 9 from at least three independent experiments. PS, protein stain.