C75, a known inhibitor of fatty acid synthase is postulated to cause significant weight loss through decreased hypothalamic neuropeptide Y (NPY) production. Peripherally, C75, an ␣-methylene-␥-butyrolactone, reduces adipose tissue and fatty liver, despite high levels of malonyl-CoA. To investigate this paradox, we studied the effect of C75 on fatty acid oxidation and energy production in diet-induced obese (DIO) mice and cellular models. Whole-animal calorimetry showed that C75-treated DIO mice had a 50% greater weight loss, and a 32.9% increased production of energy because of fatty acid oxidation, compared with paired-fed controls. Etomoxir, an inhibitor of carnitine O-palmitoyltransferase-1 (CPT-1), reversed the increased energy expenditure in DIO mice by inhibiting fatty acid oxidation. C75 treatment of rodent adipocytes and hepatocytes and human breast cancer cells increased fatty acid oxidation and ATP levels by increasing CPT-1 activity, even in the presence of elevated concentrations of malonyl-CoA. Studies in human cancer cells showed that C75 competed with malonyl-CoA, as measured by CPT-1 activity assays. Thus, C75 acts both centrally to reduce food intake and peripherally to increase fatty acid oxidation, leading to rapid and profound weight loss, loss of adipose mass, and resolution of fatty liver. The pharmacological stimulation of CPT-1 activity is a novel finding. The dual action of the C75 class of compounds as fatty acid synthase inhibitors and CPT-1 agonists has therapeutic implications in the treatment of obesity and type II diabetes. C 75 and its family of ␣-methylene-␥-butyrolactones are known inhibitors of fatty acid synthase (FAS) (1). Treatment of mice with C75 alters the expression of hypothalamic neuropeptides, leading to reversible inanition and weight loss (2-4). In addition to its central action, C75 treatment caused changes in peripheral tissues, including inhibition of hepatic fatty acid synthesis, reduction of fatty liver, diminished adipose tissue mass, and high levels of malonyl-CoA (2, 4, 5).Malonyl-CoA, in addition to its role as a substrate for FAS, is pivotal to energy regulation through its reversible inhibition of O-carnitine palmitoyltransferase-1 (CPT-1) (6). CPT-1 catalyzes the esterification of long-chain acyl-CoAs to L-carnitine for transport into mitochondria for fatty acid oxidation. During energy excess, the increased malonyl-CoA generated for fatty acid synthesis inhibits CPT-1 activity, preventing the oxidation of newly formed fatty acids bound for energy storage. During starvation, malonyl-CoA levels fall to permit the oxidation of fatty acids for energy. When FAS is pharmacologically inhibited, malonyl-CoA levels abruptly rise (2, 5).Taken together, the peripheral effects of C75 gave rise to a paradox. How could there be a selective reduction in adipocyte mass and fatty liver in the setting of elevated levels of malonylCoA as a result of FAS inhibition? We hypothesized that C75 might have additional effects on fatty acid oxidation and CPT-1 activity.Specifical...

Fatty acid synthase (FAS), the enzyme responsible for the de novo synthesis of fatty acids, is highly expressed in ovarian cancers and most common human carcinomas. Inhibition of FAS and activation of AMP-activated protein kinase (AMPK) have been shown to be cytotoxic to human cancer cells in vitro and in vivo. In this report, we explore the cytotoxic mechanism of action of FAS inhibition and show that C93, a synthetic FAS inhibitor, increases the AMP/ATP ratio, activating AMPK in SKOV3 human ovarian cancer cells, which leads to cytotoxicity. As a physiologic consequence of AMPK activation, acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis, was phosphorylated and inhibited whereas glucose oxidation was increased. Despite these attempts to conserve energy, the AMP/ATP ratio increased with worsening cellular redox status. Pretreatment of SKOV3 cells with compound C, an AMPK inhibitor, substantially rescued the cells from C93 cytotoxicity, indicating its dependence on AMPK activation. 5-(Tetradecyloxy)-2-furoic acid, an ACC inhibitor, did not activate AMPK despite inhibiting fatty acid synthesis pathway activity and was not significantly cytotoxic to SKOV3 cells. This indicates that substrate accumulation from FAS inhibition triggering AMPK activation, not end-product depletion of fatty acids, is likely responsible for AMPK activation. C93 also exhibited significant antitumor activity and apoptosis against SKOV3 xenografts in athymic mice without significant weight loss or cytotoxicity to proliferating cellular compartments such as bone marrow, gastrointestinal tract, or skin. Thus, pharmacologic FAS inhibition selectively activates AMPK in ovarian cancer cells, inducing cytotoxicity while sparing most normal human tissues from the pleiotropic effects of AMPK activation. [Cancer Res 2007;67(7):2964-71]

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