Dopaminergic (DA) signaling governs the control of complex behaviors, and its deregulation has been implicated in a wide range of diseases. Here we demonstrate that inactivation of the Fto gene, encoding a nucleic acid demethylase, impairs dopamine receptor type 2 (D2R) and type 3 (D3R) (collectively, 'D2-like receptor')-dependent control of neuronal activity and behavioral responses. Conventional and DA neuron-specific Fto knockout mice show attenuated activation of G protein-coupled inwardly-rectifying potassium (GIRK) channel conductance by cocaine and quinpirole. Impaired D2-like receptor-mediated autoinhibition results in attenuated quinpirole-mediated reduction of locomotion and an enhanced sensitivity to the locomotor- and reward-stimulatory actions of cocaine. Analysis of global N(6)-methyladenosine (m(6)A) modification of mRNAs using methylated RNA immunoprecipitation coupled with next-generation sequencing in the midbrain and striatum of Fto-deficient mice revealed increased adenosine methylation in a subset of mRNAs important for neuronal signaling, including many in the DA signaling pathway. Several proteins encoded by these mRNAs had altered expression levels. Collectively, FTO regulates the demethylation of specific mRNAs in vivo, and this activity relates to the control of DA transmission.

Adipose triglyceride lipase (ATGL) is a recently described adipose-enriched protein with triglyceride-specific lipase activity. ATGL shares the greatest sequence homology with adiponutrin, a nutritionally regulated protein of unclear biological function. Here we present a functional analysis of ATGL and adiponutrin and describe their regulation by insulin. Retroviral-mediated overexpression of ATGL in 3T3-L1 adipocytes increased basal and isoproterenol-stimulated glycerol and nonesterified fatty acid (NEFA) release, whereas siRNA-mediated knockdown of ATGL had the opposite effect. In contrast, siRNA-mediated knockdown of adiponutrin in 3T3-L1 adipocytes had no effect on glycerol or NEFA release. In mice, both ATGL and adiponutrin are nutritionally regulated in adipose tissue, with ATGL being upregulated and adiponutrin being downregulated by fasting. In 3T3-L1 adipocytes, insulin decreased ATGL and increased adiponutrin expression in a dose-and time-dependent manner, suggesting that insulin directly mediates this nutritional regulation. In addition, adipose expression of ATGL was increased by insulin deficiency and decreased by insulin replacement in streptozotocininduced diabetic mice and was increased in fat-specific insulin receptor knockout mice, whereas adiponutrin showed the opposite pattern. These data suggest that murine ATGL but not adiponutrin contributes to net adipocyte lipolysis and that ATGL and adiponutrin are oppositely regulated by insulin both in vitro and in vivo. Diabetes 55: 148 -157, 2006 A dipose tissue triglycerides are the predominant form of energy storage in animals. The ability to store and release this energy in response to variable energy availability is advantageous to survival and requires a carefully regulated balance between triglyceride synthesis and hydrolysis. Dysregulation of these processes may result in metabolic disorders, such as obesity and lipodystrophy, which are associated with dyslipidemia, insulin resistance, and overt diabetes. Hence understanding the regulatory mechanisms underlying the storage and mobilization of triglycerides is essential to understanding the pathophysiology of obesity, diabetes, and related metabolic disorders.Hormone-sensitive lipase (HSL) has traditionally been considered the key lipolytic enzyme in adipocytes (1-4). Lipolytic hormones such as catecholamines stimulate lipolysis primarily via cAMP-mediated activation of protein kinase (PK) A. PKA then phosphorylates HSL and perilipin A. Phosphorylation of perilipin A alleviates the barrier function of this protein and prompts its active participation in the lipolytic process. Phosphorylation of HSL results in translocation of HSL from the cytosol to the lipid droplet where it catalyzes the hydrolysis of tri-, di-, and monoglycerides; cholesteryl esters; and other substrates. Insulin acutely inhibits lipolysis, at least in part, via inhibition of the above cAMP-dependent pathway by PKBdependent phosphorylation and activation of phosphodiesterase 3B, which in turn lowers cAMP levels (5). How...

SF-1-expressing neurons of the ventromedial hypothalamus (VMH) control energy homeostasis, but the role of insulin action in these cells remains undefined. We show that insulin activates PI3-kinase (PI3k) signaling in SF-1 neurons and reduces firing frequency in these cells via activation of KATP-channels. These effects are abrogated in mice with insulin receptor (IR) deficiency restricted to SF-1 neurons (SF-1ΔIR-mice). While body weight and glucose homeostasis remain unaltered in SF-1ΔIR-mice under normal chow diet, they exhibit protection from diet-induced leptin resistance, weight gain, adiposity and impaired glucose tolerance. High-fat feeding activates PI3k signaling in SF-1 neurons of control mice, and this response is attenuated in the VMH of SF-1ΔIR-mice. Mimicking diet-induced overactivation of PI3k signaling by disruption of the PIP3-phosphatase PTEN leads to increased body weight and hyperphagia under normal chow diet. Collectively, our experiments reveal a critical role for HFD-induced, insulin-dependent PI3k activation in VMH neurons to control energy homeostasis.

Dopaminergic midbrain neurons integrate signals on food palatability and food-associated reward into the complex control of energy homeostasis. To define the role of insulin receptor (IR) signaling in this circuitry, we inactivated IR signaling in tyrosine hydroxylase (Th)-expressing cells of mice (IR(ΔTh)). IR inactivation in Th-expressing cells of mice resulted in increased body weight, increased fat mass, and hyperphagia. While insulin acutely stimulated firing frequency in 50% of dopaminergic VTA/SN neurons, this response was abolished in IR(ΔTh) mice. Moreover, these mice exhibited an altered response to cocaine under food-restricted conditions. Taken together, these data provide in vivo evidence for a critical role of insulin signaling in catecholaminergic neurons to control food intake and energy homeostasis.

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When rats are given access to a running-wheel in combination with food restriction, they will become hyperactive and decrease their food intake, a paradoxical phenomenon known as activity-based anorexia (ABA). Little is known about the regulation of the hypothalamic neuropeptides that are involved in the regulation of food intake and energy balance during the development of ABA. Therefore, rats were killed during the development of ABA, before they entered a state of severe starvation. Neuropeptide mRNA expression levels were analysed using quantitative real-time PCR on punches of separate hypothalamic nuclei. As is expected in a state of negative energy balance, expression levels of agouti-related protein (AgRP) and neuropeptide Y (NPY) were increased 5-fold in the arcuate nucleus (ARC) of food-restricted running ABA rats vs 2-fold in sedentary food-restricted controls. The co-regulated expression of AgRP and NPY strongly correlated with relative body weight and white adipose tissue mass. Arcuate expression of pro-opiomelanocortin (POMC) and cocaine-and amphetamine-regulated transcript (CART) was reduced 2-fold in the ABA group. In second-order neurons of the lateral hypothalamic area (LHA), melanin-concentrating hormone (MCH) mRNA expression was upregulated 2-fold in food-restricted running rats, but not in food-restricted sedentary controls. Prepro-orexin, CART and corticotropin-releasing hormone expression levels in the LHA and the paraventricular nucleus (PVN) were unchanged in both food-restricted groups. From this study it was concluded that during the development of ABA, neuropeptides in first-order neurons in the ARC and MCH in the LHA are regulated in an adequate response to negative energy balance, whereas expression levels of the other studied neuropeptides in secondary neurons of the LHA and PVN are unchanged and are probably regulated by factors other than energy status alone.

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Hyperactivity in anorexia nervosa is difficult to control and negatively impacts outcome. Hyperactivity is a key driving force to starvation in an animal model named activity-based anorexia (ABA). Recent research has started unraveling what mechanisms underlie this hyperactivity. Besides a general increase in locomotor activity that may be an expression of foraging behavior and involves frontal brain regions, the increased locomotor activity expressed before food is presented (food anticipatory behavior or FAA) involves hypothalamic neural circuits. Ghrelin plays a role in FAA, whereas decreased leptin signaling is involved in both aspects of increased locomotor activity. We hypothesize that increased ghrelin and decreased leptin signaling drive the activity of dopamine neurons in the ventral tegmental area. In anorexia nervosa patients, this altered activity of the dopamine system may be involved not only in hyperactivity but also in aberrant cognitive processing related to food.