Pharmacological glycerol-3-phosphate acyltransferase inhibition decreases food intake and adiposity and increases insulin sensitivity in diet-induced obesity.

Storage of excess calories as triglycerides is central to obesity and its associated disorders. Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial step in acylglyceride syntheses, including triglyceride synthesis. We utilized a novel small-molecule GPAT inhibitor, FSG67, to investigate metabolic consequences of systemic pharmacological GPAT inhibition in lean and diet-induced obese (DIO) mice. FSG67 administered intraperitoneally decreased body weight and energy intake, without producing conditioned taste aversion. Daily FSG67 (5 mg/kg, 15.3 µmol/kg) produced gradual 12% weight loss in DIO mice beyond that due to transient 9- to 10-day hypophagia (6% weight loss in pair-fed controls). Continued FSG67 maintained the weight loss despite return to baseline energy intake. Weight was lost specifically from fat mass. Indirect calorimetry showed partial protection by FSG67 against decreased rates of oxygen consumption seen with hypophagia. Despite low respiratory exchange ratio due to a high-fat diet, FSG67-treated mice showed further decreased respiratory exchange ratio, beyond pair-fed controls, indicating enhanced fat oxidation. Chronic FSG67 increased glucose tolerance and insulin sensitivity in DIO mice. Chronic FSG67 decreased gene expression for lipogenic enzymes in white adipose tissue and liver and decreased lipid accumulation in white adipose, brown adipose, and liver tissues without signs of damage. RT-PCR showed decreased gene expression for orexigenic hypothalamic neuropeptides AgRP or NPY after acute and chronic systemic FSG67. FSG67 given intracerebroventricularly (100 and 320 nmol icv) produced 24-h weight loss and feeding suppression, indicating contributions from direct central nervous system sites of action. Together, these data point to GPAT as a new potential therapeutic target for the management of obesity and its comorbidities.

AMPK in the brain: its roles in energy balance and neuroprotection.

Adenosine monophosphate-activated protein kinase (AMPK) senses metabolic stress and integrates diverse physiological signals to restore energy balance. Multiple functions are indicated for AMPK in the CNS. While all neurons sense their own energy status, some integrate neuro-humoral signals to assess organismal energy balance. A variety of disease states may involve AMPK, so determining the underlying mechanisms is important. We review the impact of altered AMPK activity under physiological (hunger, satiety) and pathophysiological (stroke) conditions, as well as therapeutic manipulations of AMPK that may improve energy balance.

Physiological glucose is critical for optimized neuronal viability and AMPK responsiveness in vitro.

Understanding the mechanisms that govern neuronal responses to oxidative and metabolic stress is essential for therapeutic intervention. In vitro modeling is an important approach for these studies, as the metabolic environment influences neuronal responses. Surprisingly, most neuronal culture methods employ conditions that are non-physiological, especially with regards to glucose concentrations, which often exceed 20mM. This concentration is a significant departure from physiological glucose levels, and even several-fold greater than that seen during severe hyperglycemia. The goal of this study was to establish a physiological neuronal culture system that will facilitate the study of neuronal energy metabolism and responses to metabolic stress. We demonstrate that the metabolic environment during preparation, plating, and maintenance of cultures affects neuronal viability and the response of neuronal pathways to changes in energy balance.

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.

Fatty acid synthase inhibition activates AMP-activated protein kinase in SKOV3 human ovarian cancer cells.

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.

Fatty acid metabolism, the central nervous system, and feeding.

A potential role for fatty acid metabolism in the regulation of energy balance in the brain or in the periphery has been considered only recently. Fatty acid synthase (FAS) catalyzes the synthesis of long-chain fatty acids, whereas the breakdown of fatty acids by beta-oxidation is regulated by carnitine palmitoyltransferase-1, the rate-limiting enzyme for the entry of fatty acids into the mitochondria for oxidation. While the question of the physiological role of fatty acid metabolism remains to be resolved, studies indicate that inhibition of FAS or stimulation of carnitine palmitoyltransferase-1 using cerulenin or synthetic FAS inhibitors reduces food intake and incurs profound and reversible weight loss. Several hypotheses regarding the mechanisms by which these small molecules mediate their effects have been entertained. Centrally, these compounds alter the expression of hypothalamic neuropeptides, generally reducing the expression of orexigenic peptides. Whether through central, peripheral, or combined central and peripheral mechanisms, these compounds also increase energy consumption to augment weight loss. In vitro and in vivo studies indicate that at least part of C75's effects is mediated by modulation of adenosine monophosphate-activated protein kinase, a member of an energy-sensing kinase family. These compounds, with chronic treatment, also alter gene expression peripherally to favor a state of enhanced energy consumption. Together, these effects raise the possibility that pharmacological alterations in fatty acid synthesis/degradation may serve as a target for obesity therapeutics.

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.

Fatty acid metabolism as a target for obesity treatment.

Although metabolites and energy balance have long been known to play roles in the regulation of food intake, the potential role of fatty acid metabolism in this process has been considered only recently. Fatty acid synthase (FAS) catalyzes the condensation of acetyl-CoA and malonyl-CoA to generate long-chain fatty acids in the cytoplasm, while the breakdown of fatty acids (beta-oxidation) occurs in mitochondria and is regulated by carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting step for the entry of fatty acids into the mitochondria. Inhibition of FAS using cerulenin or synthetic FAS inhibitors such as C75 reduces food intake and induces profound reversible weight loss. Subsequent studies reveal that C75 also stimulates CPT-1 and increases beta-oxidation. Hypotheses as to the mechanisms by which C75 and cerulenin mediate their effects have been proposed. Centrally, these compounds alter the expression profiles of feeding-related neuropeptides, often inhibiting the expression of orexigenic peptides. Whether through centrally mediated or peripheral mechanisms, C75 also increases energy consumption, which contributes to weight loss. In vitro and in vivo studies demonstrate that at least part of C75's effects is mediated by modulation of AMP-activated protein kinase (AMPK), a known peripheral energy-sensing kinase. Collectively, these data suggest a role for fatty acid metabolism in the perception and regulation of energy balance.

Pharmacological inhibition of AMP-activated protein kinase provides neuroprotection in stroke.

The restoration of energy balance during ischemia is critical to cellular survival; however, relatively little is known concerning the regulation of neuronal metabolic pathways in response to central nervous system ischemia. AMP-activated protein kinase (AMPK), a master sensor of energy balance in peripheral tissues, is phosphorylated and activated when energy balance is low. We investigated whether AMPK might also modulate neuronal energy homeostasis during ischemia. We utilized two model systems of ischemia, middle cerebral artery occlusion in vivo and oxygen-glucose deprivation in vitro, to delineate changes in AMPK activity incurred from a metabolic stress. AMPK is highly expressed in cortical and hippocampal neurons under both normal and ischemic conditions. AMPK activity, as assessed by phosphorylation status, is increased following both middle cerebral artery occlusion and oxygen-glucose deprivation. Pharmacological inhibition of AMPK by either C75, a known modulator of neuronal ATP levels, or compound C reduced stroke damage. In contrast, activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside exacerbated damage. Mice deficient in neuronal nitric-oxide synthase demonstrated a decrease in both stroke damage and AMPK activation compared with wild type, suggesting a possible interaction between NO and AMPK activation in stroke. These data demonstrate a role for AMPK in the response of neurons during metabolic stress and suggest that in ischemia the activation of AMPK is deleterious. The ability to manipulate pharmacologically neuronal energy balance during ischemia represents an innovative approach to neuroprotection.

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.

Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment.

We previously demonstrated that C75, a specific and potent inhibitor of fatty acid synthase (FAS), reduced food intake and decreased body weight in mice. In the present study, we determined that these effects were not due to conditioned taste aversion. To investigate the mechanism of C75 action, we examined FAS brain expression. FAS was expressed in a number of brain regions, including arcuate and paraventricular nuclei (PVN) within regions that comprise the arcuate-PVN pathway in mouse and human. Although C75 and fasting significantly downregulated liver FAS, FAS levels remained high in hypothalamus, indicating that FAS levels were regulated differently in brain from those in liver. Double fluorescence in situ for FAS and neuropeptide Y (NPY) showed that FAS co-localized with NPY in neurons in the arcuate nucleus. NPY immnuoreactivity after C75 treatment was decreased in axon terminals that innervate the PVN and lateral hypothalamus. Collectively, these results demonstrate that FAS is present and active in neurons and suggests that C75 may alter food intake via interactions within the arcuate-PVN pathway mediated by NPY.

C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity.

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 alpha-methylene-gamma-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.