Phenethyl Isothiocyanate (PEITC) interaction with Keap1 activates the Nrf2 pathway and inhibits lipid accumulation in adipocytes

Phenethyl isothiocyanate (PEITC) has been recognized for its potential effects in various human diseases. However, the impact of PEITC on adipocyte differentiation and its underlying molecular mechanisms is not well understood. This study investigates the effects of PEITC on adipocyte differentiation and elucidates the molecular mechanisms involved in Nrf2 activation. The effects of PEITC on adipocyte differentiation were assessed in C3H10T1/2 and 3T3-L1 cells. Nrf2-induced effects by PEITC were examined in Nrf2 knockout (KO) MEF and Keap1 KO H1299 cells. The interaction between PEITC and Keap1 was evaluated using thermal shift assays and Co-immunoprecipitation experiments. Reconstitution of cysteine mutants of Keap1 in Keap1 KO cells was used to elucidate a critical amino acid for the PEITC-induced Nrf2 stabilization. The initial stages of adipogenesis were crucial for PEITC's antiadipogenic effects in C3H10T1/2 and 3T3-L1 cells. PEITC increased Nrf2 protein expression, but this induction was absent in Keap1 KO cells. Thermal shift assays with the purified BTB domain of Keap1 confirmed a direct interaction with PEITC. Re-expression of Keap1 in Keap1 KO cells showed that the cysteine residue at position 151 is essential for PEITC-induced Nrf2 expression and the disruption of the Nrf2-Keap1 complex. PEITC was found to activate Nrf2-mediated gene expression and inhibit adipocyte differentiation, at least partially, through Nrf2-dependent mechanisms. This study confirms the antiadipogenic effects of PEITC. Mechanistic investigations demonstrate that PEITC interacts with Keap1 and that the cysteine residue (C151) of Keap1 is critical for PEITC's effects on Nrf2 activation.