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.

Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake.

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptin's Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptin's anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptin-mediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptin's signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptin's anorectic signaling cascade.

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.

Heme oxygenase-2 protects against glutathione depletion-induced neuronal apoptosis mediated by bilirubin and cyclic GMP.

Heme oxygenase (HO) enzymes catalyze the breakdown of heme to iron, carbon monoxide (CO), and biliverdin, which is rapidly converted to bilirubin. HO-2 has been implicated in protection against oxidative stress, ischemia, and traumatic brain injury. The neuroprotective effects of HO-2 have been attributed to the generation of bilirubin, which is an important radical scavenger. However, the mechanism by which HO-2 provides protection is unclear. We utilized the olfactory system as a model to define the roles of HO-2 in glutathione depletion-induced oxidative injury, since olfactory receptor neurons (ORNs) express high levels of HO isoforms. We demonstrated that L-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione biosynthesis, lowered glutathione levels and induced apoptosis of ORNs. Despite the presence of HO-1 in ORNs, HO-2 null animals displayed increased levels of neuronal death after BSO treatment compared to wild type mice. Levels of bilirubin and cGMP were also reduced in HO-2 null mice. Primary cultures of ORNs confirmed that the neuroprotective role of HO-2 was mediated by bilirubin and cGMP. Taken together, these results suggest that HO-2 plays a major role in neuroprotection from oxidative stress, an effect that is mediated by cGMP and bilirubin.

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons.

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.

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.

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.

The localization of neuronal nitric oxide synthase may influence its role in neuronal precursor proliferation and synaptic maintenance.

Neuronal nitric oxide synthase (nNOS) is implicated in some developmental processes, including neuronal survival, differentiation, and precursor proliferation. To define the roles of nNOS in neuronal development, we utilized the olfactory system as a model. We hypothesized that the role of nNOS may be influenced by its localization. nNOS expression was developmentally regulated in the olfactory system. During early postnatal development, nNOS was expressed in developing neurons in the olfactory epithelium (OE), while in the adult its expression was restricted to periglomerular (PG) cells in the olfactory bulb (OB). At postnatal week 1 (P1W), loss of nNOS due to targeted gene deletion resulted in a decrease in immature neurons in the OE due to decreased proliferation of neuronal precursors. While the pool of neuronal precursors and neurogenesis normalized in the nNOS null mouse by P6W, there was an overgrowth of mitral or tufted cells dendrites and a decreased number of active synapses in the OB. Cyclic GMP (cGMP) immunostaining was reduced in the OE and in the glomeruli of the OB at early postnatal and adult ages, respectively. Our results suggest that nNOS appears necessary for neurogenesis in the OE during early postnatal development and for glomerular organization in the OB in the adult. Thus, the location of nNOS, either within cell bodies or perisynaptically, may influence its developmental role.

Expression of MeCP2 in olfactory receptor neurons is developmentally regulated and occurs before synaptogenesis.

Rett syndrome, a neurodevelopmental disorder hypothesized to be due to defective neuronal maturation, is a result of mutations in the mecp2 gene encoding the transcriptional repressor methyl-CpG binding protein (MeCP2). We utilized the olfactory system, which displays postnatal neurogenesis, as a model to investigate MeCP2 expression during development and after injury. MeCP2 expression increased postnatally, localizing to mature olfactory receptor neurons (ORNs) and sustentacular supporting cells. The timing of MeCP2 expression was defined by using detergent ablation (to remove the ORNs) and unilateral olfactory bulbectomy (to remove the ORN target), both of which increase neurogenesis. MeCP2 expression in the ORNs reached prelesioning levels as cells matured after ablation, whereas expression was not completely restored after bulbectomy, in which functional synaptogenesis cannot occur. Thus, MeCP2 expression correlates with the maturational state of ORNs, and precedes synaptogenesis. Identifying the time window of MeCP2 expression should help further clarify the biological defects in Rett syndrome.

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin, respectively.

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.