Rodent models of obesity induced by consuming high-fat diet (HFD) are characterized by inflammation both in peripheral tissues and in hypothalamic areas critical for energy homeostasis. Here we report that unlike inflammation in peripheral tissues, which develops as a consequence of obesity, hypothalamic inflammatory signaling was evident in both rats and mice within 1 to 3 days of HFD onset, prior to substantial weight gain. Furthermore, both reactive gliosis and markers suggestive of neuron injury were evident in the hypothalamic arcuate nucleus of rats and mice within the first week of HFD feeding. Although these responses temporarily subsided, suggesting that neuroprotective mechanisms may initially limit the damage, with continued HFD feeding, inflammation and gliosis returned permanently to the mediobasal hypothalamus. Consistent with these data in rodents, we found evidence of increased gliosis in the mediobasal hypothalamus of obese humans, as assessed by MRI. These findings collectively suggest that, in both humans and rodent models, obesity is associated with neuronal injury in a brain area crucial for body weight control.

SUMMARY Mitofusin 2 (Mfn2) plays critical roles in both mitochondrial fusion and the establishment of mitochondria-endoplasmic reticulum (ER) interactions. Hypothalamic ER stress has emerged as a causative factor for the development of leptin resistance, but the underlying mechanisms are largely unknown. Here we show that mitochondria-ER contacts in anorexigenic pro-opiomelanocortin (POMC) neurons in the hypothalamus are decreased in diet-induced obesity. POMC-specific ablation of Mfn2 resulted in loss of mitochondria-ER contacts, defective POMC processing, ER stress-induced leptin resistance, hyperphagia, reduced energy expenditure and obesity. Pharmacological relieve of hypothalamic ER stress reversed these metabolic alterations. Our data establishes Mfn2 in POMC neurons as an essential regulator of systemic energy balance by fine-tuning the mitochondrial-ER axis homeostasis and function. This previously unrecognized role for Mfn2 argues for a crucial involvement in mediating ER stress-induced leptin resistance.

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.

The sirtuins are a family of highly conserved NAD+-dependent deacetylases that act as cellular sensors to detect energy availability and modulate metabolic processes. Two sirtuins that are central to the control of metabolic processes are mammalian sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), which are localized to the nucleus and mitochondria, respectively. Both are activated by high NAD+ levels, a condition caused by low cellular energy status. By deacetylating a variety of proteins that induce catabolic processes while inhibiting anabolic processes, SIRT1 and SIRT3 coordinately increase cellular energy stores and ultimately maintain cellular energy homeostasis. Defects in the pathways controlled by SIRT1 and SIRT3 are known to result in various metabolic disorders. Consequently, activation of sirtuins by genetic or pharmacological means can elicit multiple metabolic benefits that protect mice from diet-induced obesity, type 2 diabetes, and nonalcoholic fatty liver disease.

The hypothalamic paraventricular nucleus (PVN) functions as a center to integrate various neuronal activities for regulating feeding behavior. Nesfatin-1, a recently discovered anorectic molecule, is localized in the PVN. However, the anorectic neural pathway of nesfatin-1 remains unknown. Here we show that central injection of nesfatin-1 activates the PVN and brain stem nucleus tractus solitarius (NTS). In the PVN, nesfatin-1 targets both magnocellular and parvocellular oxytocin neurons and nesfatin-1 neurons themselves and stimulates oxytocin release. Immunoelectron micrographs reveal nesfatin-1 specifically in the secretory vesicles of PVN neurons, and immunoneutralization against endogenous nesfatin-1 suppresses oxytocin release in the PVN, suggesting paracrine/autocrine actions of nesfatin-1. Nesfatin-1-induced anorexia is abolished by an oxytocin receptor antagonist. Moreover, oxytocin terminals are closely associated with and oxytocin activates pro-opiomelanocortin neurons in the NTS. Oxytocin induces melanocortin-dependent anorexia in leptin-resistant Zucker-fatty rats. The present results reveal the nesfatin-1-operative oxytocinergic signaling in the PVN that triggers leptin-independent melanocortin-mediated anorexia.

Roles for hypothalamic reactive oxygen species (ROS) in the modulation of circuit activity of the melanocortin system were proposed1,2,. Here we show that suppression of ROS diminished pro-opiomelanocortin (POMC) cell activation and promoted the activity of neuropeptide Y- (NPY)/agouti related peptide- (AgRP) neurons and feeding, whereas ROS activated POMC neurons and reduced feeding. ROS in POMC neurons were positively correlated with leptin levels in lean and ob/ob animals a relationship diminished in diet-induced obese (DIO) mice. High fat feeding resulted hypothalamic proliferation of peroxisomes and elevated PPARγ mRNA levels. Peroxisome proliferation in POMC neurons by the PPARγ agonist, rosiglitazone, decreased ROS levels and increased food intake in lean mice on high fat diet. Suppression of peroxisome proliferation in the hypothalamus by the PPAR antagonist, GW9662, increased ROS and c-fos expression in POMC neurons, reversed high fat feeding-triggered elevated NPY/AgRP and low POMC neuronal firing, and, resulted in decreased feeding of DIO mice. Finally, central administration of ROS alone increased c-fos and pStat3 expression in POMC neurons and reduced feeding of DIO animals. These observations unmask a previously unknown hypothalamic cellular event associated with peroxisomes and ROS in the central regulation of energy metabolism in states of leptin resistance.

We have shown that synaptic re-organization of hypothalamic feeding circuits in response to metabolic shifts involves astrocytes, cells that can directly respond to the metabolic hormone, leptin, in vitro. It is not known whether the role of glia cells in hypothalamic synaptic adaptions is active or passive. Here we show that leptin receptors are expressed in hypothalamic astrocytes and that conditional, adult deletion of leptin receptors in astrocytes leads to altered glial morphology, decreased glial coverage and elevated synaptic inputs onto pro-opiomelanocortin (POMC)- and Agouti-related protein (AgRP)-producing neurons. Leptin-induced suppression of feeding was diminished, while rebound feeding after fasting or ghrelin administration was elevated in mice with astrocyte-specific leptin receptor deficiency. These data unmask an active role of glial cells in the initiation of hypothalamic synaptic plasticity and neuroendocrine control of feeding by leptin.

The fasting-activated longevity protein sirtuin 1 (SirT1, ref. 1 ) promotes gluconeogenesis in part, by increasing transcription of the key gluconeogenic genes pepck1 and g6pase 2,3 , through deacetylating ). In contrast, signal transducer and activator of transcription 3 (STAT3) inhibits glucose production by suppressing expression of these genes 5,6 . It is not known whether the inhibition of gluconeogenesis by STAT3 is controlled by metabolic regulation. Here we show that STAT3 phosphorylation and function in the liver were tightly regulated by the nutritional status of an animal, through SirT1-mediated deacetylation of key STAT3 lysine sites. The importance of the SirT1-STAT3 pathway in the regulation of gluconeogenesis was verified in STAT3-deficient mice in which the dynamic regulation of gluconeogenic genes by nutritional status was disrupted. Our results reveal a new nutrient sensing pathway through which SirT1 suppresses the inhibitory effect of STAT3, while activating the stimulatory effect of PGC-1α and FOXO1 on gluconeogenesis, thus ensuring maximal activation of gluconeogenic gene transcription. The connection between acetylation and phosphorylation of STAT3 implies that STAT3 may have an important role in other cellular processes that involve SirT1.The transcription factor STAT3 participates in various critical cellular processes 7 . In the liver, STAT3 is known to suppress expression of the transcriptional co-activator of gluconeogenesis PGC-1α and to suppress gluconeogenic genes. Ectopic expression of STAT3 in leptin receptor mutant (lepr -/-) mice reduces PGC-1α transcription and reverses diabetes. This effect of STAT3 [8][9][10] . However, the functional significance of STAT3 acetylation remains ill-defined, and its relationship with STAT3 tyrosine phosphorylation remains unclear. NIH Public AccessWe hypothesized that STAT3 acetylation regulates physiological processes by mediating changes in the STAT3 phosphorylation status. We found that STAT3 acetylation was decreased after a 24-h fast and increased after feeding in the livers of C57BL/6J mice. STAT3 tyrosine phosphorylation directly correlated with the level of STAT3 acetylation, indicating that the reversible acetyl-modifications are functionally relevant (Fig. 1a). The observation that both acetylation and phosphorylation of STAT3 were evident in fed, but dramatically reduced in fasted, animals suggests that STAT3 acetylation and phosphorylation are actively downregulated on fasting. Overall, these observations signify that cellular metabolic status regulates STAT3 function in the liver, presumably owing to the sensitivity of the liver to the overall nutritional status of the organism 11 .SirT1 can be induced to promote gluconeogenesis 11,12 under conditions of fasting. Therefore, we asked whether SirT1 affects fed/fast-regulated STAT3 acetylation and phosphorylation. We injected the SirT1 inhibitor EX527, which has shown increased potency and specificity for SirT1 (ref. 13 ), into C57Bl/6J mice. EX527 increased acetylation and...