Increasing fatty acid oxidation remodels the hypothalamic neurometabolome to mitigate stress and inflammation.

Modification of hypothalamic fatty acid (FA) metabolism can improve energy homeostasis and prevent hyperphagia and excessive weight gain in diet-induced obesity (DIO) from a diet high in saturated fatty acids. We have shown previously that C75, a stimulator of carnitine palmitoyl transferase-1 (CPT-1) and fatty acid oxidation (FAOx), exerts at least some of its hypophagic effects via neuronal mechanisms in the hypothalamus. In the present work, we characterized the effects of C75 and another anorexigenic compound, the glycerol-3-phosphate acyltransferase (GPAT) inhibitor FSG67, on FA metabolism, metabolomics profiles, and metabolic stress responses in cultured hypothalamic neurons and hypothalamic neuronal cell lines during lipid excess with palmitate. Both compounds enhanced palmitate oxidation, increased ATP, and inactivated AMP-activated protein kinase (AMPK) in hypothalamic neurons in vitro. Lipidomics and untargeted metabolomics revealed that enhanced catabolism of FA decreased palmitate availability and prevented the production of fatty acylglycerols, ceramides, and cholesterol esters, lipids that are associated with lipotoxicity-provoked metabolic stress. This improved metabolic signature was accompanied by increased levels of reactive oxygen species (ROS), and yet favorable changes in oxidative stress, overt ER stress, and inflammation. We propose that enhancing FAOx in hypothalamic neurons exposed to excess lipids promotes metabolic remodeling that reduces local inflammatory and cell stress responses. This shift would restore mitochondrial function such that increased FAOx can produce hypothalamic neuronal ATP and lead to decreased food intake and body weight to improve systemic metabolism.

Pharmacological stimulation of brain carnitine palmitoyl-transferase-1 decreases food intake and body weight.

Inhibition of brain carnitine palmitoyl-transferase-1 (CPT-1) is reported to decrease food intake and body weight in rats. Yet, the fatty acid synthase (FAS) inhibitor and CPT-1 stimulator C75 produces hypophagia and weight loss when given to rodents intracerebroventricularly (icv). Thus roles and relative contributions of altered brain CPT-1 activity and fatty acid oxidation in these phenomena remain unclarified. We administered compounds that target FAS or CPT-1 to mice by single icv bolus and examined acute and prolonged effects on feeding and body weight. C75 decreased food intake rapidly and potently at all doses (1-56 nmol) and dose dependently inhibited intake on day 1. Dose-dependent weight loss on day 1 persisted through 4 days of postinjection monitoring. The FAS inhibitor cerulenin produced dose-dependent (560 nmol) hypophagia for 1 day, weight loss for 2 days, and weight regain to vehicle control by day 3. The CPT-1 inhibitor etomoxir (32, 320 nmol) did not alter overall day 1 feeding. However, etomoxir attenuated the hypophagia produced by C75, indicating that CPT-1 stimulation is important for C75's effect. A novel compound, C89b, was characterized in vitro as a selective stimulator of CPT-1 that does not affect fatty acid synthesis. C89b (100, 320 nmol) decreased feeding in mice for 3 days and produced persistent weight loss for 6 days without producing conditioned taste aversion. Similarly, intraperitoneal administration decreased feeding and body weight without producing conditioned taste aversion. These results suggest a role for brain CPT-1 in the regulation of energy balance and implicate CPT-1 stimulation as a pharmacological approach to weight loss.

The connections between C75 and obesity drug-target pathways.

Obesity and its attendant disorders, such as Type II diabetes, have reached epidemic proportions in the USA, and their prevalence is increasing globally. C75 is a small-molecule inhibitor of fatty acid synthase (FAS) and a stimulator of carnitine palmitoyl 1 activity, which causes profound weight loss in mice. Although C75 is not a compound that is destined for human drug development, it has provided two potential pathways to target in obesity therapy: fatty acid synthesis and fatty acid oxidation. In this article, we discuss the latest data challenging the relationship between fatty acid synthase inhibition and C75-induced anorexia.

C75 alters central and peripheral gene expression to reduce food intake and increase energy expenditure.

C75, a synthetic inhibitor of fatty acid synthase (FAS), causes anorexia and profound weight loss in lean and genetically obese mice. C75 also acts as a stimulator of carnitine palmitoyltransferase-1 to induce fatty acid oxidation. To approximate human obesity, we used a 2-wk C75 treatment model for diet-induced obese (DIO) mice to investigate the central and peripheral effects of C75 on gene expression. C75 treatment decreased food intake, increased energy expenditure, and reduced body weight more effectively in DIO than in lean mice. Analysis of the gene expression changes in hypothalamus demonstrated that the reduced food intake in C75-treated DIO mice might be mediated by inhibition of orexigenic neuropeptide expression and induction of anorexigenic neuropeptide expression. Gene expression changes in peripheral tissues indicated that C75 increased energy expenditure by the induction of genes involved in fatty acid oxidation. C75 also inhibited the expression of genes in peripheral tissues responsible for fatty acid synthesis and accumulation. The patterns of the changes in central and peripheral gene expression that occur with C75 treatment provide mechanisms to explain the reduced food intake and increased energy expenditure observed with C75.

C75, a fatty acid synthase inhibitor, reduces food intake via hypothalamic AMP-activated protein kinase.

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-beta-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 alpha subunit (pAMPKalpha) in the hypothalamus, even in fasted mice that had elevated hypothalamic pAMPKalpha levels. Furthermore, AICAR reversed both the C75-induced anorexia and the decrease in hypothalamic pAMPKalpha levels. C75 elevated hypothalamic neuronal ATP levels, which may contribute to the mechanism by which C75 decreased AMPK activity. C75 reduced the levels of pAMPKalpha 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.

C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

A new model ELISA, based on two monoclonal antibodies, for quantification of fatty acid synthase.

A new model ELISA, based on two monoclonal antibodies, was developed for the quantification of fatty acid synthase (FAS). In this sandwich assay, a monoclonal antibody M6 was used as a capture on Nunc MaxiSorp ELISA/EIA Modules and another monoclonal antibody M3, labeled with biotin, was used as a detection antibody. More than 10 molecules of biotin were labeled on the anti-FAS monoclonal antibody using modified biotinylation conditions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/mL. Recoveries were 98.54-121.95%, averaging 106.05%. The average FAS concentration obtained from the total 55 healthy volunteers blood was 4.07 +/- 1.81 ng/mL, 4.25 +/- 2.14 ng/mL in women (n = 37) and 3.70 +/- 0.74 ng/mL in men (n = 18). When compared with the previously developed polyclonal-monoclonal ELISA, a different pattern of FAS levels was observed in the supernatant of two cultured breast cancer cell lines in a time course study and there was no linear correlation between the two assays using 215 human blood samples. Thus, this new model FAS-ELISA could be used as an independent assay in measuring clinical samples. In summary, this monoclonal-monoclonal FAS-ELISA is sensitive, accurate, and precise in quantification of fatty acid synthase and has potential as a complementary tool in testing clinical samples.