FIGURE 2.

Inhibition of lipolysis with the general lipase inhibitor orlistat blunts the activation of AMPK by agents that increase cAMP. 3T3-L1 adipocytes were treated with IBMX (0.5 mm), isoproterenol (Iso, 10 μm), or forskolin (20 μm) for 1 h, following a 4-h preincubation period in serum-free DMEM containing 0.5% fatty acid-free BSA with/without orlistat (100 μm). Lipolysis was assessed by measuring the release of glycerol (A) or FFA (B) into the incubation media and the activation of AMPK by immunoblots (IB) of phospho-AMPK and phospho-ACC (C) as described in the legend of Fig. 1. Densitometric analyses of P-AMPK (D) and P-ACC (E) are shown. N.D., nondetectable. Immunoblots shown are representative of nine samples obtained in three independent experiments. Results in A, B, D, and E are expressed as means ± S.E. (n = 9) and were obtained from three independent experiments. Significantly different from control group: ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001; significantly different from orlistat-treated cells: ++, p < 0.01; +++, p < 0.001.