With the escalation of obesity-related disease, there is great interest in defining the mechanisms that control appetite and body weight. We have identified a link between anabolic energy metabolism and appetite control. Both systemic and intracerebroventricular treatment of mice with fatty acid synthase (FAS) inhibitors (cerulenin and a synthetic compound C75) led to inhibition of feeding and dramatic weight loss. C75 inhibited expression of the prophagic signal neuropeptide Y in the hypothalamus and acted in a leptin-independent manner that appears to be mediated by malonyl-coenzyme A. Thus, FAS may represent an important link in feeding regulation and may be a potential therapeutic target.

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Fatty acid synthase (FAS), the sole mammalian enzyme capable of de novo fatty acid synthesis, is highly expressed in most human carcinomas. FAS is associated with poor prognosis in breast and prostate cancer, is elaborated into the blood of cancer patients, and its inhibition is selectively cytotoxic to human cancer cells. Thus, FAS and fatty acid metabolism in cancer has become a focus for the potential diagnosis and treatment of cancer. (Cancer Res 2006; 66(12): 5977-80)

In 1989, we reported the immunologic identification of a prognostic molecule in the tumor cells of breast cancer patients with a poor prognosis for recurrence and death due to their disease (1). This prognostic factor was statistically independent ofimportant clinical parameters including tumor size, lymph node involvement, and assessment of estrogen and progesterone receptors; subsequent studies also showed it to be independent of other prognostic molecules including c-erb-B2 and cathepsin D (2). Tumors marked by this prognostic molecule were nearly 4 times more likely to recur and metastasize than tumors not so marked, representing a prognostic power as strong as the presence of cancer in the axillary lymph nodes ofpatients with T1 or T2 primaries (1-3).We

There is no known treatment for fatty liver, a ubiquitous cause of chronic liver disease. However, because it is associated with hyperinsulinemia and insulin-resistance, insulin-sensitizing agents might be beneficial. To evaluate this possibility, insulin-resistant ob/ob mice with fatty livers were treated with metformin, an agent that improves hepatic insulin-resistance. Metformin improved fatty liver disease, reversing hepatomegaly, steatosis and aminotransferase abnormalities. The therapeutic mechanism likely involves inhibited hepatic expression of tumor necrosis factor (TNF) alpha and TNF-inducible factors that promote hepatic lipid accumulation and ATP depletion. These findings suggest a mechanism of action for metformin and identify novel therapeutic targets in insulin-resistant states.

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...

Compared to normal human tissues, many common human cancers, including carcinoma of the colon, prostate, ovary, breast, and endometrium, express high levels of fatty acid synthase (FAS, EC ), the primary enzyme responsible for the synthesis of fatty acids. This differential expression of FAS between normal tissues and cancer has led to the notion that FAS is a target for anticancer drug development. Recent studies with C75, an inhibitor of fatty acid synthesis, have shown significant antitumor activity with concomitant inhibition of fatty acid synthesis in tumor tissue and normal liver. Importantly, histopathological analysis of normal tissues after C75 treatment showed no adverse effects on proliferating cellular compartments, such as bone marrow, gastrointestinal tract, skin, or lymphoid tissues. In this study, we describe the de novo synthesis of C75 based on the known mechanism of action of cerulenin and the theoretical reaction intermediates of the beta-ketoacyl synthase moiety of FAS. In addition, we demonstrate that C75 is a synthetic, chemically stable inhibitor of FAS. C75 inhibits purified mammalian FAS with characteristics of a slow-binding inhibitor and also inhibits fatty acid synthesis in human cancer cells. Treatment of human breast cancer cells with [5-(3)H]C75 demonstrates that C75 reacts preferentially with FAS in whole cells. Therefore, we have shown that the primary mechanism of the antitumor activity of C75 is likely mediated through its interaction with, and inhibition of, FAS. This development will enable the in vivo study of FAS inhibition in human cancer and other metabolic diseases.

Energy homeostasis and feeding are regulated by the central nervous system. C75, a fatty acid synthase (FAS) inhibitor, causes weight loss and anorexia, implying a novel central nervous system pathway(s) for sensing energy balance. AMP-activated protein kinase (AMPK), a sensor of peripheral energy balance, is phosphorylated and activated when energy sources are low. Here, we identify a role for hypothalamic AMPK in the regulation of feeding behavior and in mediating the anorexic effects of C75. 5-Aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR), an activator of AMPK, increased food intake, whereas compound C, an inhibitor of AMPK, decreased food intake. C75 rapidly reduced the level of the phosphorylated AMPK ␣ subunit (pAMPK␣) in the hypothalamus, even in fasted mice that had elevated hypothalamic pAMPK␣ levels. Furthermore, AICAR reversed both the C75-induced anorexia and the decrease in hypothalamic pAMPK␣ levels. C75 elevated hypothalamic neuronal ATP levels, which may contribute to the mechanism by which C75 decreased AMPK activity. C75 reduced the levels of pAMPK␣ and phosphorylated cAMP response element-binding protein (pCREB) in the arcuate nucleus neurons of the hypothalamus, suggesting a mechanism for the reduction in NPY expression seen with C75 treatment. These data indicate that modulation of FAS activity in the hypothalamus can alter energy perception via AMPK, which functions as a physiological energy sensor in the hypothalamus.Despite significant advances in the understanding of appetite and satiety at molecular levels (1-3), practical therapies for weight loss remain elusive. We and others (4 -8) have demonstrated that C75, a synthetic fatty acid synthase (FAS) 1 inhibitor, caused profound weight loss and anorexia in lean, dietinduced obese (DIO) and genetically obese (ob/ob) mice. In addition to FAS inhibition, C75 also stimulates carnitine palmitoyltransferase-1 (CPT-1) activity, increasing fatty acid oxidation and ATP levels (8). Since enzymes of the fatty acid metabolic pathways are highly expressed in hypothalamic neurons that regulate feeding behavior (9), we hypothesize that C75-induced alterations in fatty acid metabolism may affect neuronal energy flux, which could signal a change in energy status, leading to changes in feeding behavior.AMPK (AMP-activated protein kinase) is activated by metabolic stresses such as nutrient starvation (10) and ischemiahypoxia (11) and by physiological processes such as vigorous exercise (12, 13). Specifically, increases in the AMP/ATP ratio, decreases in cellular pH, and increases in the creatine/phosphocreatine ratio are known to activate AMPK via allosteric activation of AMPK by AMP and by phosphorylation of AMPK by AMPKK (14 -19). Once activated, AMPK switches off ATPconsuming biosynthetic pathways such as fatty acid synthesis and switches on ATP-generating metabolic pathways such as fatty acid oxidation to preserve ATP levels (20, 21). The central roles of AMPK in both energy sensing and the control of fatty acid metabolism (16,22) and its r...