Eur J Nutr (2006) 45 : 159– 164 DOI 10.1007/s00394-005-0576-5 ■ Summary Background Genis- tein increases CPT1A, a rate-limit- ing enzyme in the β-oxidation pathway, enzyme activity by in- creasing CPT1A transcription in HepG2 cells and, consequently, suppresses high fat induced obesity in C57BL/6J mice. Genistein and daidzein are the most abundant isoflavones in soy. Aim of study To investigate the effect of co-treat- ment of genistein and L-carnitine on CPT1A enzyme activity and to determine whether daidzein also increases CPT1A activity and to es- Received: 7 June 2004 Accepted: 5 July 2005 Published online: 20 December 2005 E. S. Shin · S.Y. Cho · E. H. Lee · S. J. Lee · I. S. Chang · Dr. T. R. Lee () BioResearch, R&D Center AmorePacific Corporation 314-1, Bora-ri, Giheung-eup Yongin-si, Gyeonggi-do, 449-729, Korea Tel.: +82-31/280-5961 Fax: +82-31/281-8392 E-Mail: trlee@amorepacific.com tablish a cell line that can be used to screen chemicals to regulate CPT1A transcription. Methods The enzyme activities of CPT1A were determined after HepG2 cells were incubated with 10 µM genistein or 10 µM daidzein or 1 mM L-carni- tine or in combination with 10 µM genistein and 1 mM L-carnitine or in combination with 10 µM daidzein and 1 mM L-carnitine. The mRNA expression levels of CPT1A were determined by real time PCR method after HepG2 cells were incubated with 10 µM genis- tein or 10 µM daidzein. A suggested CPT1A promoter region was cloned from human genomic DNA and the CPT1A promoter-lu- ciferase reporter gene construct was made, and the promoter-re- porter gene construct was trans- fected into human hepatoma cell line Huh7. Results The enzyme ac- tivity of CPT1A was at least 2.3- fold higher in L-carnitine and genistein co-treated HepG2 cells than either single-agent treated cells. Daidzein also significantly in- creased the mRNA expression of CPT1A as well as the enzyme activ- ity of CPT1A. A stable Huh7 cell line, which was selected after Huh7 cells were transfected with CPT1A promoter luciferase reporter gene construct, was characterized by confirming that luciferase activity of the cell line can be regulated by genistein and daidzein as well as clofibrate, a well-known CPT1A mRNA up-regulating drug. Conclu- sions Genistein and daidzein can up-regulate CPT1A enzyme activ- ity through up-regulation of CPT1A transcription. Co-treatment of L-carnitine and genistein addi- tively increases CPT1A enzyme ac- tivity in HepG2 cells. A stable Huh7 cell line transfected with the CPT1A promoter luciferase re- porter gene was established and characterized. ■ Key words carnitine palmitoyltransferase 1A – genistein – daidzein – L-carnitine – β-oxidation ORIGINAL CONTRIBUTION Eui Seok Shin Si Young Cho Eun Hee Lee Sang Jun Lee Ih Seop Chang Tae Ryong Lee Positive regulation of hepatic carnitine palmitoyl transferase 1A (CPT1A) activities by soy isoflavones and L-carnitine Introduction Genistein and daidzein are the most abundant isoflavones in soy and derived from the hydrolysis of their glycoside forms present in soy products. They regulate the mRNA level of genes involved in cholesterol metabolism, such as hydroxymethylglutaryl-CoA (HMG-CoA) reductase, low-density lipoprotein (LDL) receptor in HepG2 cell [1]. However, the effects of isoflavones on β-oxidation have not been known al- though the β-oxidation is an important pathway for fatty acid metabolism. Carnitine palmitoyltransferase 1 (CPT1) is at the outer mitochondrial membrane and catalyzes the long- chain fatty acid translocation into the mitochondrial EJN 576