Figure 11.

Effect of phosphorylation of HSL/LD on Rimonabant-induced lipolysis in the cell-free system. LD prepared from untreated (open, filled, hatched bars; LD) or Rimonabant (10 µM, 3 h, 37 °C)-treated (RIM-LD) or CP55.490-treated (50 µM; CP-LD) primary rat adipocytes, which had been metabolically labelled with NBD-FA, were incubated (30 min, 30 °C) with ATP in the presence (pLD) or absence (LD) of PKA catalytic subunit. Recombinant human HSL was incubated (30 min, 4 °C) with ATP in the presence (pHSL) or absence (HSL) of PKA catalytic subunit. After termination of the reactions and recovery of the LD and HSL, they were reconstituted as indicated and then incubated (90 min, 30 °C) in the presence or absence of 10 µM Rimonabant (RIM) or 50 µM CP55.490 (CP). After extraction of the total incubation mixtures with chloroform/methanol/HCl, the organic phases were analyzed by TLC. The amounts of NBD-FA lipolytically released from the NBD-FA-labelled AG1–4 were quantitatively evaluated by fluorescence imaging (mean ± SD of incubations in quadriplicate and TLC analysis in duplicate. NBD-FA released in the “HSL + LD” (absence of Rimonabant during cell-free lipolysis and adipocyte incubation, respectively) was set at 100. Values from corresponding reactions lacking exogenously added HSL were subtracted in each case in order to correct for HSL copurifying with the LD. Thus, the lipolysis activity depicted as NBD-FA released relies on the exogenously added recombinant human HSL rather than endogenous LD-associated rat HSL. * p ≤ 0.01, ^# p ≤ 0.05; significant differences are indicated only for physiologically relevant comparisons as discussed in the text.