4,4-Dimethylsterols Reduces Fat Accumulation via Inhibiting Fatty Acid Amide Hydrolase In Vitro and In Vivo.

4,4-Dimethylsterols constitute a unique class of phytosterols responsible for regulating endogenous cannabinoid system (ECS) functions. However, precise mechanism through which 4,4-dimethylsterols affect fat metabolism and the linkage to the ECS remain unresolved. In this study, we identified that 4,4-dimethylsterols, distinct from 4-demethseterols, act as inhibitors of fatty acid amide hydrolases (FAAHs) both in vivo and in vitro. Genetic ablation of FAAHs () abolishes the effects of 4,4-dimethylsterols on fat accumulation and locomotion behavior in a model. We confirmed that dietary intervention with 4,4-dimethylsterols in a high-fat diet (HFD) mouse model leads to a significant reduction in body weight (>11.28%) with improved lipid profiles in the liver and adipose tissues and increased fecal triacylglycerol excretion. Untargeted and targeted metabolomics further verified that 4,4-dimethylsterols influence unsaturated fatty acid biosynthesis and elevate oleoyl ethanolamine levels in the intestine. We propose a potential molecular mechanism in which 4,4-dimethylsterols engage in binding interactions with the catalytic pocket (Ser241) of FAAH-1 protein due to the shielded polarity, arising from the presence of 2 additional methyl groups (CH). Consequently, 4,4-dimethylsterols represent an unexplored class of beneficial phytosterols that coordinate with FAAH-1 activity to reduce fat accumulation, which offers new insight into intervention strategies for treating diet-induced obesity.