Table 1.

Effects of orlistat on proteins of the endocannabinoid system and triglyceride lipase

	Source of protein	IC50 or Ki* value (10−6m)
aDiacylglycerol lipase α	human enzyme expressed in COS cells	0.06 ± 0.02
bMonoglyceride lipase	COS cells	> 25
cN-acylphosphatidylethanolamine-selective phospholipase D	rat enzyme expressed in HEK-293 cells	10 ± 1
dFatty acid amide hydrolase	rat brain	> 25
eTriglyceride lipase	rat liver	1.0 ± 0.2
fCB1 cannabinoid receptor	human receptor expressed in COS cells	4.0 ± 0.8*
gCB2 cannabinoid receptor	human receptor expressed in COS cells	> 25*

Means ± s.e.m. of n = 4 experiments.

^aDiacylglycerol lipase α activity was studied in membranes (100 μg protein) using 1-[¹⁴C]oleoyl-2-arachidonoyl-glycerol (1 mCi mmol⁻¹, 2.5 × 10⁻⁵m) as substrate (see Bisogno et al. 2003).

^bMonoglyceride lipase activity was studied in the cytosol derived from the 10 000 g fraction of homogenates (100 μg protein) using 2-[³H]arachidonoyl-glycerol (1.0 mCi mmol⁻¹, 2.5 × 10⁻⁵m) as substrate (see Bisogno et al. 2003). Wild-type COS cells express high amounts of monoglyceride lipase mRNA and the indicated fraction exhibits the highest monoglyceride lipase activity (T. Bisogno & V. Di Marzo, unpublished observations).

^cN-Acylphosphatidylethanolamine-selective phospholipase D activity was studied in membrane fractions from confluent HEK-293 cells transiently transfected with this enzyme using phosphatidylethanolamine-N-[³H]-arachidonoyl (200 Ci mmol⁻¹, 10⁻⁴m) as substrate (see Okamoto et al. 2004). Lipids were extracted with two volumes of chloroform/methanol (2/1; v/v). The extracts were purified by thin-layer chromatography (TLC) on silica plates eluted with the organic phase from a mixture of isooctane/ethylacetate/water/acetic acid (50/110/100/20; v/v/v/v). Radioactivity in TLC bands corresponding to anandamide or arachidonic acid was determined.

^dFatty acid amide hydrolase activity was studied in membranes prepared from rat brain using [¹⁴C]anandamide (5 mCi mmol⁻¹, 5 × 10⁻⁶m) as substrate (see Cascio et al. 2004).

^eTriglyceride lipase was studied in debris-free homogenates of rat liver (100 μg protein) using 1,2,3-[¹⁴C]oleoyl-glycerol as substrate (100 mCi mmol⁻¹, 2.5 × 10⁻⁵m). After extraction and TLC, radioactivity in TLC bands corresponding to [¹⁴C]oleic acid was determined.

^f,gBinding assays on membranes from COS cells transfected with either recombinant human CB₁ or CB₂ receptors were performed as previously described (see Appendino et al. 2005).