Prevalence of loss-of-function FTO mutations in lean and obese individuals.

OBJECTIVE: Single nucleotide polymorphisms (SNPs) in intron 1 of fat mass- and obesity-associated gene (FTO) are strongly associated with human adiposity, whereas Fto(-/-) mice are lean and Fto(+/-) mice are resistant to diet-induced obesity. We aimed to determine whether FTO mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure-function relationships within FTO. RESEARCH DESIGN AND METHODS: We sequenced all coding exons of FTO in 1,433 severely obese and 1,433 lean individuals. We studied the enzymatic activity of selected nonsynonymous variants. RESULTS: We identified 33 heterozygous nonsynonymous variants in lean (2.3%) and 35 in obese (2.4%) individuals, with 8 mutations unique to the obese and 11 unique to the lean. Two novel mutations replace absolutely conserved residues: R322Q in the catalytic domain and R96H in the predicted substrate recognition lid. R322Q was unable to catalyze the conversion of 2-oxoglutarate to succinate in the presence or absence of 3-methylthymidine. R96H retained some basal activity, which was not enhanced by 3-methylthymidine. However, both were found in lean and obese individuals. CONCLUSIONS: Heterozygous, loss-of-function mutations in FTO exist but are found in both lean and obese subjects. Although intron 1 SNPs are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that FTO has a role in energy balance, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese. Functional analyses of FTO mutations have given novel insights into structure-function relationships in this enzyme.

Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations.

FTO is a nuclear protein belonging to the AlkB-related non-haem iron- and 2-oxoglutarate-dependent dioxygenase family. Although polymorphisms within the first intron of the FTO gene have been associated with obesity, the physiological role of FTO remains unknown. Here we show that a R316Q mutation, inactivating FTO enzymatic activity, is responsible for an autosomal-recessive lethal syndrome. Cultured skin fibroblasts from affected subjects showed impaired proliferation and accelerated senescence. These findings indicate that FTO is essential for normal development of the central nervous and cardiovascular systems in human and establish that a mutation in a human member of the AlkB-related dioxygenase family results in a severe polymalformation syndrome.

Fatty acid synthase inhibitors modulate energy balance via mammalian target of rapamycin complex 1 signaling in the central nervous system.

OBJECTIVE: Evidence links the hypothalamic fatty acid synthase (FAS) pathway to the regulation of food intake and body weight. This includes pharmacological inhibitors that potently reduce feeding and body weight. The mammalian target of rapamycin (mTOR) is an intracellular fuel sensor whose activity in the hypothalamus is also linked to the regulation of energy balance. The purpose of these experiments was to determine whether hypothalamic mTOR complex 1 (mTORC1) signaling is involved in mediating the effects of FAS inhibitors. RESEARCH DESIGN AND METHODS: We measured the hypothalamic phosphorylation of two downstream targets of mTORC1, S6 kinase 1 (S6K1) and S6 ribosomal protein (S6), after administration of the FAS inhibitors C75 and cerulenin in rats. We evaluated food intake in response to FAS inhibitors in rats pretreated with the mTOR inhibitor rapamycin and in mice lacking functional S6K1 (S6K1(-/-)). Food intake and phosphorylation of S6K1 and S6 were also determined after C75 injection in rats maintained on a ketogenic diet. RESULTS: C75 and cerulenin increased phosphorylation of S6K1 and S6, and their anorexic action was reduced in rapamycin-treated rats and in S6K1(-/-) mice. Consistent with our previous findings, C75 was ineffective at reducing caloric intake in ketotic rats. Under ketosis, C75 was also less efficient at stimulating mTORC1 signaling. CONCLUSIONS: These findings collectively indicate an important interaction between the FAS and mTORC1 pathways in the central nervous system for regulating energy balance, possibly via modulation of neuronal glucose utilization.

The role of CNS fuel sensing in energy and glucose regulation.

Individual cells must carefully regulate their energy flux to ensure nutrient levels are adequate to maintain normal cellular activity. The same principle holds in multicellular organisms. Thus, for mammals to perform necessary physiological functions, sufficient nutrients need to be available. It is more complex, however, to understand how the energy status of different cells impacts on the overall energy balance of the entire organism. We propose that the central nervous system is the critical organ for the coordination of intracellular metabolic processes that are essential to guarantee energy homeostasis at the organismal level. In particular, we suggest that in specific hypothalamic neurons, evolutionarily conserved fuel sensors, such as adenosine monophosphate-activated protein kinase and mammalian target of rapamycin (mTOR), integrate sensory input from nutrients, including those derived from recently ingested food or those that are stored in adipose tissue, to regulate effector pathways responsible for fuel intake and utilization. The corollary to this hypothesis is that dysregulation of these fuel-sensing mechanisms in the brain may contribute to metabolic dysregulation underlying diseases, such as obesity and type 2 diabetes.

Hypothalamic mTOR signaling regulates food intake.

The mammalian Target of Rapamycin (mTOR) protein is a serine-threonine kinase that regulates cell-cycle progression and growth by sensing changes in energy status. We demonstrated that mTOR signaling plays a role in the brain mechanisms that respond to nutrient availability, regulating energy balance. In the rat, mTOR signaling is controlled by energy status in specific regions of the hypothalamus and colocalizes with neuropeptide Y and proopiomelanocortin neurons in the arcuate nucleus. Central administration of leucine increases hypothalamic mTOR signaling and decreases food intake and body weight. The hormone leptin increases hypothalamic mTOR activity, and the inhibition of mTOR signaling blunts leptin's anorectic effect. Thus, mTOR is a cellular fuel sensor whose hypothalamic activity is directly tied to the regulation of energy intake.

Central administration of melanin-concentrating hormone increases alcohol and sucrose/quinine intake in rats.

BACKGROUND: Alcohol is a caloric compound that can contribute to energy intake. Therefore, peptides that regulate energy balance likely modify the motivation to consume alcohol. Melanin-concentrating hormone (MCH) regulates energy homeostasis and has been implicated in other behaviors that impact alcohol consumption (i.e., anxiety, fluid balance, and reward). We tested the hypothesis that MCH would decrease the motivation to consume alcohol secondarily to reducing anxiety. METHODS: Rats were trained to drink 10% ethanol or an isocaloric concentration of sucrose with use of a sucrose-fading technique. MCH (1, 5, or 10 microg) or its saline vehicle was administered into the third cerebral ventricle (i3vt), and intake of ethanol or sucrose and chow was assessed for 2 hr. Alcohol-naïve rats were evaluated in an elevated plus maze after i3vt MCH (10 microg), neuropeptide Y, or saline administration. RESULTS: Contrary to the hypothesis, MCH dose-dependently increased alcohol intake: saline = 0.7 +/- 0.1 g/kg, 1 microg MCH = 1.0 +/- 0.1 g/kg, 5 microg MCH = 1.2 +/- 0.1 g/kg, and 10 microg MCH = 1.8 +/- 0.3 g/kg (p < 0.01), and this was true whether water was simultaneously available or not. MCH also significantly increased sucrose intake (saline = 1.0 +/- 0.3 g/kg, 10 mug MCH = 1.4 +/- 0.5 g/kg; p < 0.05). MCH had no effect on time spent in the open arms (54.3 +/- 11.5 sec) relative to saline (58.2 +/- 23.8 sec), whereas neuropeptide Y, a known anxiolytic, increased time spent on the open arms (119.2 +/- 22 sec, p < 0.05). CONCLUSIONS: We conclude that MCH nonspecifically increases ingestive behavior. Furthermore, MCH had no apparent effect on anxiety. The ability of MCH to increase alcohol and/or sucrose intake may be explained by the effect of MCH on energy balance and/or reward processes.

Mechanisms of oleoylethanolamide-induced changes in feeding behavior and motor activity.

Oleoylethanolamide (OEA), a lipid synthesized in the intestine, reduces food intake and stimulates lipolysis through peroxisome proliferator-activated receptor-alpha. OEA also activates transient receptor potential vanilloid type 1 (TRPV1) in vitro. Because the anorexigenic effect of OEA is associated with delayed feeding onset and reduced locomotion, we examined whether intraperitoneal administration of OEA results in nonspecific behavioral effects that contribute to the anorexia in rats. Moreover, we determined whether circulating levels of other gut hormones are modulated by OEA and whether CCK is involved in OEA-induced anorexia. Our results indicate that OEA reduces food intake without causing a conditioned taste aversion or reducing sodium appetite. It also failed to induce a conditioned place aversion. However, OEA induced changes in posture and reduced spontaneous activity in the open field. This likely underlies the reduced heat expenditure and sodium consumption observed after OEA injection, which disappeared within 1 h. The effects of OEA on motor activity were similar to those of the TRPV1 agonist capsaicin and were also observed with the peroxisome proliferator-activated receptor-alpha agonist Wy-14643. Plasma levels of ghrelin, peptide YY, glucagon-like peptide 1, and apolipoprotein A-IV were not changed by OEA. Finally, antagonism of CCK-1 receptors did not affect OEA-induced anorexia. These results suggest that OEA suppresses feeding without causing visceral illness and that neither ghrelin, peptide YY, glucagon-like peptide 1, apolipoprotein A-IV, nor CCK plays a critical role in this effect. Despite that OEA-induced anorexia is unlikely to be due to impaired motor activity, our data raise a cautionary note in how specific behavioral and metabolic effects of OEA should be interpreted.

Mother to infant or infant to mother? Reciprocal regulation of responsiveness to stress in rodents and the implications for humans.

Optimal early development in most species is dependent upon a stable relationship between the mother and her infant. The research described here focuses on the reciprocal nature of this dyad in rodents and humans, with respect to the regulation of responsiveness to stress in both mother and offspring. Dietary influences are critical not only to regulate infant growth but also to modulate the response of the neuroendocrine system to stress and, possibly, to influence some aspects of brain development. In particular, we discuss the role of leptin, a protein produced in the adipose tissue and present in maternal milk, that reduces responses to stress in the infant. We suggest that leptin acts on both central (hypothalamus and hippocampus) and peripheral (pituitary, adrenal gland) targets in the infant to reduce exposure to glucocorticoids and enhance hippocampal development during a sensitive period of brain development. There is also evidence to support the reverse regulatory influence, in which maternal state is profoundly affected by stimulation from the young. During the period of lactation, mothers exhibit lower neuroendocrine and behavioural responses to several types of stressors, except possibly those representing a threat to the infant. This ability to "filter" relevant from irrelevant stimuli while caring for their young might be viewed as adaptive for the mother-infant dyad, and the inability to filter adequately stressful stimuli could at least in part be associated with the development of postpartum depression.

Leptin regulates appetite-related neuropeptides in the hypothalamus of developing rats without affecting food intake.

Leptin regulates food intake in adult mammals by stimulating hypothalamic anorexigenic pathways and inhibiting orexigenic ones. In developing rodents, fat stores are low, yet circulating leptin levels are high and do not appear to regulate food intake. We determined whether two appetite-related neuropeptides [neuropeptide Y (NPY) and proopiomelanocortin (POMC)] and food intake behavior are sensitive to leptin [3 mg/kg body weight (BW), ip] in neonates. We measured the effects of 1) acute leptin administration (3 mg/kg BW, ip, 3 h before testing) on food intake on postnatal day (PND) 5, 8, and 10; and 2) chronic leptin treatment (3 mg/kg BW, ip, daily PND3-PND10) on BW gain and fat pads weight on PND10. In addition to hypothalamic POMC and NPY expression, we determined the expression of suppressor of cytokine signaling-3, all subtypes of leptin receptors, and corticotropin-releasing factor receptor-2 mRNA in PND10 pups receiving either an acute (PND10) or a chronic (PND 3-10) leptin (3 mg/kg BW, ip) or vehicle treatment. Brains were removed 30 or 120 min after the last injection. Acute leptin administration did not affect food intake at any age tested. Chronic leptin treatment did not change BW but decreased fat pad weight significantly. In the arcuate nucleus (ARC), acute leptin increased SOCS-3 and POMC mRNA levels, but decreased NPY mRNA levels in the rostral part of ARC. Chronic leptin down-regulated all subtypes of leptin receptors mRNA and decreased NPY mRNA levels in the caudal ARC but had no further effect on POMC expression. Chronic leptin increased corticotropin-releasing factor receptor-2 mRNA levels in the ventromedial hypothalamus. We conclude that despite adult-like effects of leptin on POMC, NPY, and CRFR-2 expression in neonates, leptin does not regulate food intake during early development.

Comparisons of behavioral and neurochemical characteristics between WKY, WKHA, and Wistar rat strains.

WKHA rats constitute a recombinant inbred rat strain derived by phenotypic selection of the progeny of hybrid F2 crosses between SHR and WKY rats. WKHA are normotensive and show some features of hyperactivity and of hyper-reactivity to stress, but their utility as model of attention deficit/hyperactivity disorder (ADHD) has not yet been settled. To address these questions, we performed behavioral and neurochemical evaluations of WKHA, and compared them to both WKY and Wistar (WIS) rats. In locomotor activity tests, the respective scores for each strain were WKY