NF-κB Activation in Hypothalamic Pro-opiomelanocortin Neurons Is Essential in Illness- and Leptin-induced Anorexia*

Anorexia and weight loss are prevalent in infectious diseases. To investigate the molecular mechanisms underlying these phenomena, we established animal models of infection-associated anorexia by administrating bacterial and viral products, lipopolysaccharide (LPS) and human immunodeficiency virus-1 transactivator protein (Tat). In these models, we found that the nuclear factor-κB (NF-κB), a pivotal transcription factor for inflammation-related proteins, was activated in the hypothalamus. In parallel, administration of LPS and Tat increased hypothalamic pro-inflammatory cytokine production, which was abrogated by inhibition of hypothalamic NF-κB. In vitro, NF-κB activation directly stimulated the transcriptional activity of pro-opiomelanocortin (POMC), a precursor of anorexigenic melanocortin, and mediated the stimulatory effects of LPS, Tat, and pro-inflammatory cytokines on POMC transcription, implying the involvement of NF-κB in controlling feeding behavior. Consistently, hypothalamic injection of LPS and Tat caused a significant reduction in food intake and body weight, which was prevented by blockade of NF-κB and melanocortin. Furthermore, disruption of IκB kinase-β, an upstream kinase of NF-κB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-κB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-κB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-κB also serves as a downstream signaling pathway of leptin.

with infectious diseases (1,2). Infection-associated anorexia is not merely the consequence of fever and general weakness (3), but it appears to occur due to an alteration in the motivation to eat (4). The suppression of food intake during acute infection may constitute a host defense mechanism as poor nutritional states might limit the proliferation of infectious organisms (5). However, a prolonged reduction in food intake will deplete body fat and protein reserves, resulting in malnutrition, impaired host immune functions, and increased morbidity and mortality (6). Therefore, elucidating the mechanism of infection-associated anorexia and weight loss should aid in improving the quality of life in patients with chronic debilitating infections.
Cumulating evidence suggests that pro-inflammatory cytokines produced in the periphery and brain play a role in infection-associated anorexia and weight loss (7). Because appetite is regulated by the central nervous system, infection-associated anorexia may be mediated via the central mechanisms. Consistent with this hypothesis, a recent study has demonstrated that central inflammatory signals are important for bacterial endotoxin lipopolysaccharide (LPS) 3 -induced anorexia (8). On the other hand, a number of studies suggest an important role of the hypothalamic melanocortin system in infection-associated anorexia (9). However, the molecular mechanisms triggering anorexia during infection are yet to be fully addressed.
Nuclear factor-B (NF-B) transcription factors were originally identified as critical regulators of genes involved in inflammation and innate immunity (10). In the quiescent state, NF-B dimers exist in an inactive form in the cytoplasm bound to the IB␣ inhibitory protein. Inflammatory stimuli activate the IB kinase (IKK) complex, which phosphorylates IB, leading to its ubiquitination and subsequent degradation. IB degradation facilitates translocation of NF-B to the nucleus, thereby regulating the transcription of genes involved in human disorders, including cancer, neurodegenerative disease, and obesity-related metabolic disease (11)(12)(13). NF-B is also involved in cancer-induced cachexia, because intratumoral injection of NF-B inhibitory oligonucleotides prevents cachexia in a mouse tumor model (14). In the present study, we present evidence that, during infection, NF-B is activated in the hypothalamus, a center for appetite and body weight regulation, and stimulates the local production of proinflammatory cytokines and anorexigenic pro-opiomelanocortin (POMC), thereby causing anorexia and weight loss.

MATERIALS AND METHODS
Animals-Male C57BL/6 mice 8 -10 weeks of age were obtained from Orient Bio Inc. (Seoul, Korea). Mice were fed standard chow (Samyang Co, Seoul, Korea) ad libitum. Animals were housed under controlled temperature (22°C) and a 12-h light-dark cycle, with light from 7:00 a.m. to 7:00 p.m. All procedures were approved by the Institutional Animal Care and Use Committee at the Asian Institute for Life Sciences.
Cell Culture-AtT-20 and SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Hypothalamic neurons were primarily cultured as previously described (15).
Preparation of Recombinant Tat Protein-Tat 1-72 cDNA was cloned and ligated into pGex4T3 vector (Amersham Biosciences). The resulting gene product was overexpressed in Escherichia coli BL21. Recombinant glutathione S-transferase-Tat was purified by affinity chromatography using glutathioneagarose 4B (Sigma).
Cannulation and Injection-Cannulation and injection into the third ventricle (ICV: 0.8 mm caudal to the bregma and 5 mm ventral to the sagittal sinus) or the mediobasal hypothalamus (intra-mediobasal hypothalamic: 1.8 mm caudal to the bregma, and 5.5 mm ventral and 0.3 mm lateral to the sagittal sinus) were performed according to a previous report (16). LPS (Sigma), Tat 1-72 , and leptin (R&D Systems) were dissolved in 0.9% saline and administered via intra-mediobasal hypothalamic-or ICV-implanted cannulae in 0.5 and 2 l of volume, respectively, at 8:00 -10:00 a.m. in mice following an overnight fast, unless otherwise indicated. For the inhibition study, Agouti-related protein (AGRP, Phoenix Pharmaceuticals), IKK inhibitory peptide (Calbiochem, #401477), and Bay 11-7085 (Sigma) were dissolved in either saline or 1% DMSO before use and injected 30 min before injection of LPS or Tat 1-72 . The doses of inhibitors used were determined from the preliminary studies with a wide range of doses and small sized groups, which alone did not significantly change food intake and body weight and blocked the effects of LPS or Tat.
Chromatin Immunoprecipitation-SH-SY5Y cells were treated with LPS (10 ng/ml) for 1 h, fixed with formaldehyde, lysed, and sonicated. Soluble chromatin was coimmunoprecipitated with anti-p50 rabbit serum (Santa Cruz Biotechnology) or an equivalent amount of rabbit immunoglobulin-␥ (IgG). After de-cross-linking of DNA, samples were subjected to PCR to amplify the first intron region of the human POMC gene (nucleotide positions ϩ2123 to ϩ2430) using the primers (5Ј-TGAAGTTTTAGCAATAGCAGC-3Ј and 5Ј-TGGTAGT-TACGTATGTCACCAA-3Ј).
Determination of mRNA Expression-Total RNA was extracted from the mediobasal hypothalamus and AtT-20 cells using TRIzol reagent (Invitrogen). The mRNA levels of proinflammatory cytokines, neuropeptides, and NF-B were determined by real-time PCR (PerkinElmer Life Sciences) or semi-quantitative reverse transcription-PCR using the primers specified in supplemental Fig. S1. Expression of each mRNA was normalized to that of glyceraldehyde-3-phosphate dehydrogenase.
Small Interfering RNA-The murine sequences of p50 (positions ϩ2624 to ϩ2643) were targeted for RNA interference (supplemental Fig. S2). AtT-20 cells were transfected with control or p50-specific small interfering RNA (100 nM) using Lipofectamine 36 h before promoter assay.
Measurement of Body Temperature-Rectal temperature was measured using a thermometer (Harvard Apparatus, Holliston, MA).
Measurement of Serum Corticosterone-Trunk blood was collected 45 min after intraperitoneal administration of LPS. Serum corticosterone levels were measured using a radioimmunoassay kit (DPC, Deerfield, IL).
Data Analysis-All data are presented as mean Ϯ S.E. Groups were compared using Student's t test or analysis of variance, followed by a post hoc least significant difference test. Significance was defined as p Ͻ 0.05.

Activation of Hypothalamic NF-B by LPS and Tat-Initially,
we investigated whether NF-B is activated in the hypothalamus during systemic infection. LPS (200 g/kg) was administered intraperitoneally in freely fed C57BL/6J mice in the early light phase and the mediobasal hypothalamus was collected to assess NF-B activity at 1 and 6 h following LPS injection. NF-B activity was determined with the EMSA using the NF-B binding consensus sequence (19). A single intraperitoneal administration of LPS led to NF-B activation in the hypothalamus at 1 h post injection (Fig. 1A). However, this effect was not sustained at 6 h (data not shown). Binding of NF-B to the corresponding DNA sequences was inhibited and supershifted in the presence of antibodies specific for the NF-B subunits, p50 and p65 (Fig. 1B), suggesting that the canonical NF-B pathway was activated (20).
NF-B is expressed in neurons and glial cells in the central nervous system. Neuronal NF-B plays an important role in both normal and pathologic conditions (20). To determine whether LPS activates NF-B in hypothalamic neurons, we performed double immunofluorescence staining using antibodies specific to p50 and the neuron marker, MAP2. We used anti-p50-antibody, because commercially available anti-p65 antibodies were all nonspecific, as previously mentioned (21). Tat, a viral protein produced by the human immunodeficiency virus, type 1, has originally been implicated in the transcription initiation and elongation of human immunodeficiency virus-encoding genes (22). In addition to its role as a transcriptional regulator, Tat is released from infected cells and acts like an extracellular cytokine (23). Extracellular Tat causes neuron toxicity through N-methyl-D-aspartic acid receptor (22). Therefore, we used Tat protein for establishment of viral infection model. Similar to LPS, biologically active Tat 1-72 increased IB phosphorylation and nuclear translocation of NF-B in primary-cultured hypothalamic neurons (supplemental Fig. S3, B and C). These findings clearly demonstrate that the Tat 1-72 protein also causes NF-B activation in hypothalamic neurons.
LPS and Tat Stimulate Hypothalamic Cytokine Production via NF-B-During systemic infection, pro-inflammatory cytokines are produced in the meninges, choroid plexus, and brain, which may provoke inflammatory responses within the central nervous system in a paracrine and autocrine manner (7). Consistent with this notion, intraperitoneal administration of LPS (200 g/kg) and Tat 1-72 (4 mg/kg) enhanced the mRNA expression of pro-inflammatory cytokines, including IL-1␤, IL-6, and tumor necrosis factor-␣ in the mediobasal hypothalamus at 3 h post-injection (Fig. 1, D and E).
To explore a role for NF-B in LPS-and Tat-induced hypothalamic cytokine production, we administered the NF-B inhibitor (24), Bay 11-7085 (500 nmol), into the third ventricle, 30 min prior to intraperitoneal administration of LPS and Tat  . ICV administration of Bay 11-7085 significantly inhibited the increase in hypothalamic cytokine expression following LPS and Tat treatment (Fig. 1, D and E). These data suggest that hypothalamic NF-B mediates LPS-and Tat-induced increase in hypothalamic cytokine expression. On the other hand, enhanced pro-inflammatory cytokine production may further amplify NF-B activation in the hypothalamus, as evidenced by the finding that treatment of IL-1␤ and tumor necrosis factor-␣ caused NF-B activation in primary cultured hypothalamic neurons (supplemental Fig. S4).
NF-B Directly Stimulates POMC Transcriptional Activity-The melanocortins, ␣-, ␤-, and ␥-melanocyte-stimulating-hormone (MSH), are produced from the precursor protein, POMC (25). Central melanocortin and its receptors (melanocortin 3 and 4 receptors, MC3R and MC4R) play an important role in the maintenance of normal food intake and body weight in rodents and humans (25). Several lines of evidence suggest that the hypothalamic melanocortin system is a potential mediator in illness-associated anorexia (26 -28). Hypothalamic ␣MSH levels are increased in pathologic conditions, such as human immunodeficiency virus infection (26). Consistently, we found that intraperitoneal administration of LPS (200 g/kg) and Tat 1-72 (4 mg/kg) increased hypothalamic POMC mRNA expression at 3 h after intraperitoneal administration ( Fig. 2A). Moreover, ICV administration of ␣MSH augments LPS-induced anorexia and cachexia, whereas the melanocortin antagonist, SHU9119, attenuates it (27). LPS-and cancer-induced anorexia and weight loss are also diminished in MC4R-null mice (28). These findings collectively suggest that hypothalamic melanocortins cause anorexia and weight loss through the MC4R during the illness. However, the mechanisms via which infectious products or pro-inflammatory cytokines activate the hypothalamic melanocortin system remain to be established.
We hypothesized that POMC is a downstream target of NF-B. Putative NF-B binding motifs were identified within the 1-kb nucleotide sequence upstream of the coding sequence (B-1) and the first intron (B-2) of the human, mouse, and rat POMC genes (Fig. 2B). To investigate the effects of NF-B on POMC transcriptional activity, we gen-erated two luciferase reporter constructs containing B-1 (nucleotides Ϫ877 to ϩ320, POMC-luc1) and B-2 (nucleotides ϩ1705 to ϩ4134, POMC-luc2) of the human POMC gene. Human p50 and/or p65 were coexpressed with POMC-luc1 or POMC-luc2 in POMC-producing murine AtT-20 cells. Expression of p50 increased POMC-luc2 activity up to ϳ8-fold (Fig. 2C), and POMC-luc1 activity only by 2-fold (supplemental Fig. S5). Interestingly, expression of p65 alone did not affect POMC-luc2 activity, whereas coexpression with p50 further enhanced POMC-luc2 activity in a dose-dependent manner (Fig. 2C). These findings, taken together with EMSA data (Fig. 1B), indicate that p50:p50 and p50:p65 dimers, but not p65:p65 dimers, may be involved in transcriptional regulation of POMC. EMSA using oligonucleotides compatible with the potential B binding sites 1 and 2 (B-1 and B-2 probes) demonstrated that the B-2 probe was better than B-1 probe for the detection of LPS-induced hypothalamic presence of p50-and p65-specific antibodies, suggesting that canonical NF-B pathway is activated. "C" represents saline-injected controls. C, triple immunohistochemistry with p50, neuron marker (MAP2), and nuclear marker (4Ј,6-diamidino-2-phenylindole (DAPI)) in mouse hypothalamic arcuate nucleus (ARC). Brains were collected at 45 min after intraperitoneal administration of saline or LPS. The intranuclear p50 immunoreactivity in MAP2-positive neurons of hypothalamic ARC were counted from three different slides per mice (n ϭ 3). p Ͻ 0.05 versus saline. D and E, hypothalamic mRNA expression of pro-inflammatory cytokines following intraperitoneal administration of saline, LPS, or Tat 1-72 with or without prior ICV administration of NF-B inhibitor (B inh.) (n ϭ 5-6 per group). Mediobasal hypothalami were collected 3 h after intraperitoneal injection. Pro-inflammatory cytokine mRNA levels were determined using semiquantitative reverse transcription-PCR and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each bar represents mean Ϯ S.E. *, p Ͻ 0.005 versus saline-injected control (C); †, p Ͻ 0.05 versus Tat alone group. (Fig. 2D). Consistently, mutation of B-2 site completely abolished p50-induced stimulation of POMC transcriptional activity, suggesting that B-2 site is critical for NF-B-mediated stimulation of POMC transcription (Fig. 2E). Finally, we confirmed that endogenous p50 binds to B-2 of the human POMC gene, using a chromatin immunoprecipitation assay. Chromatin immunoprecipitation data revealed that binding of endogenous p50 to this region was low in the basal state, but significantly increased by LPS treatment (10 ng/ml) in SH-SY5Y human neuroblastoma cells (Fig. 2F). Collectively, NF-B p50 stimulates POMC transcriptional activity through direct interactions at B-2 site.

NF-B activation
Because Tat functions as a transcriptional coactivator (22), we also examined the effects of Tat gene expression on POMC promoter activity. Expression of the Tat gene modestly increased POMC-luc2 activity in AtT-20 cells (supplemental Fig. S6A). Moreover, coexpression of Tat and p50 led to a synergistic increase in POMC transcriptional activity (supplemental Fig. S6B), suggesting that NF-B-induced POMC transcription is enhanced by endogenous Tat expression.
We additionally determined the direct effects of LPS, Tat 1-72 , and pro-inflammatory cytokines on POMC transcriptional activity. Treatment with these agents significantly stimulated POMC-luc2 transcriptional activity in both AtT-20 and SH-SY5Y cells (Fig. 2G and supplemental Fig. S6C), which was inhibited by pretreatment with a NF-B inhibitor, Bay 11-7085 (Fig. 2G). Moreover, depletion of p50 expression using a small inhibitory RNA-mediated gene-silencing system completely abolished the effects of LPS and Tat on POMC-luc2 activity (Fig. 2H). Our results strongly suggest that NF-B is an important downstream mediator in the regulation of POMC transcription by LPS, Tat, or pro-inflammatory cytokines.
Hypothalamic NF-B Mediates LPS-and Tat-induced Anorexia and Weight Loss-Consistent with a previous report (29), central administration of LPS and Tat caused a reduction in food intake and body weight (supplemental Fig. S7). Next, we investigated a role for hypothalamic NF-B in LPS-and Tatinduced anorexia and weight loss. For this, we injected an NF-B inhibitor, Bay 11-7085 (500 nmol) or IKK inhibitory peptide (30) (1 and 3 g), into the mediobasal hypothalamus 30 min prior to administration of LPS (3 ng) or Tat 1-72 (0.1 nmol). Injection of NF-B inhibitor or IKK inhibitor alone did not significantly alter food intake and body weight but blocked the effects of LPS and Tat 1-72 (Fig. 3, A-D). These findings indicate that hypothalamic NF-B activation is an important signaling event for anorexia and weight loss induced by LPS and Tat.
Inhibition of NF-B in POMC Neurons Attenuates LPS-and Tat-induced Anorexia-We further investigated the relationship of NF-B and the hypothalamic melanocortin system in feeding regulation. In line with a previous report (27), prior ICV administration of an endogenous melanocortin antagonist, AGRP (4 g), blocked the effects of LPS (10 ng) and Tat  (1 nmol) on food intake and body weight (Fig. 3, E and F). Although treatment with AGRP alone increased food intake and body weight, the blocking effect of AGRP on LPS-and Tat-induced anorexia and weight loss was much greater than that seen in AGRP-alone treatment, supporting an involvement of the central melanocortin system in illness-associated anorexia.
Given that POMC is a target gene of NF-B, we hypothesized that the central melanocortin system acts as a downstream of NF-B in infection-associated anorexia and weight loss. To define the specific role of NF-B in hypothalamic POMC neurons in illness-associate anorexia, we selectively disrupted the IKK␤ gene, a key upstream signaling pathway leading to NF-B activation (31), in POMC-producing neurons using the Cre-lox system. The Ikk␤ F/F mice (17) were crossed with Pomc-Cre transgenic mice (18) to generate POMC-specific IKK␤-depleted mice (Ikk␤ ⌬Pomc ). In Ikk␤ ⌬POMC mice, IKK␤ expression in ␣MSH-expressing neurons was significantly less than the level of Ikk␤ F/F mice (Fig. 4A). The Ikk␤ ⌬Pomc mice showed normal food intake and body weight under a standard chow diet at young adult ages compared with Pomc-Cre mice and Ikk␤ F/F mice (supplemental Fig. S8, A and B). There were no differences in plasma glucose, insulin, and leptin concentrations between Ikk␤ F/F and Ikk␤ ⌬Pomc mice (supplemental Fig. S8, C-E). Basal hypothalamic NF-B activity and POMC mRNA expression tended to be lower in Ikk␤ ⌬Pomc mice than in Ikk␤ F/F (Fig. 4, B  and C). Furthermore, LPS-induced nuclear translocation of p50 was significantly decreased in the hypothalamic POMC neurons (Fig. 4D). In Ikk␤ ⌬Pomc mice, LPS (3 ng)-and Tat (0.1 nmol)-induced reduction in food intake and weight loss was significantly attenuated, which was reversed by intrahypothalamic coadministration of ␣-MSH (Fig. 4, E-H). These findings further support the hypothesis that NF-B activation in POMC neurons plays a critical role in infection-associated anorexia and weight loss. Because melanocortins have antipyretic and anti-inflammatory effects in the central and peripheral nervous systems (26), we compared the effects of LPS on body temperature and circulating levels of anti-inflammatory hormone corticosteroid. Ikk␤ ⌬POMC mice displayed lower corticosterone levels and higher body temperatures in response to intraperitoneal LPS (200 g/kg), compared with Ikk␤ F/F mice (Fig. 4, I and J).
Leptin Causes Hypothalamic NF-B Activation-Previous studies have shown that leptin, an important anorexigenic hormone, activates NF-B in cortical neurons, microglia, and astrocytes from the brain (32)(33)(34). Therefore, we investigated whether leptin also causes hypothalamic NF-B activation. Similarly to LPS, intraperitoneal administration of leptin (3 mg/kg) significantly increased nuclear translocation of NF-B in the mediobasal hypothalamus at 1 h post-injection (Fig. 5A).
To examine if hypothalamic NF-B may serve as a downstream signaling of leptin, we administered leptin (3 g) with or without IKK inhibitor (3 g) in overnight-fasted mice via ICVimplanted cannulae. ICV administration of IKK inhibitor significantly inhibited the effects of leptin on food intake and body weight (Fig. 5, B and C), suggesting that hypothalamic NF-B activation is essential for leptin-induced anorexia and weight loss.
that NF-B mediates the effects of leptin on POMC transcription (Fig. 5, D and E).
To further confirm a role for NF-B activation of POMC neuron in leptin-induced anorexia, leptin (0.1 g) was administered into the mediobasal hypothalamus of Ikk␤ F/F and Ikk␤ ⌬POMC mice following an overnight fast. Intrahypothalamic administration of leptin induced a marked decrease in food intake and body weight in Ikk␤ F/F mice (Fig. 5, F and G). However, these effects were significantly attenuated in Ikk␤ ⌬POMC mice, suggesting that leptin-induced anorexia and weight loss depends on NF-B activation in POMC neurons.

DISCUSSION
Our findings are summarized in Fig. 5H. Hypothalamic NF-B is activated by infectious agents, LPS and Tat, or by leptin. Hypothalamic NF-B activation leads to increased production of anorexigenic cytokines and POMC in the hypothalamus. In particular, NF-B activation in hypothalamic POMC neurons is an important molecular mechanism for anorexia and weight loss as evidenced by the findings that POMC neuron-specific NF-B inhibition significantly reduced LPS, Tat, or leptin-induced anorexia and weight loss.
Although a critical role for the hypothalamic melanocortin system in illness-associated anorexia has been suggested (26 -28), it was unclear how central melanocortin system was regulated in the state of illness. In this study, we demonstrated that NF-B directly bound the human POMC gene to stimulate POMC transcription. Therefore, hypothalamic NF-B may be a missing link between illness and central melanocortin system. A, hypothalamic NF-B activation following intraperitoneal administration of leptin (3 mg/kg) and LPS (200 g/kg). Mediobasal hypothalami were collected 1 h after intraperitoneal injection following a 5-h fast. B and C, ICV administration of IKK inhibitory peptide inhibited leptin-induced anorexia and weight loss (n ϭ 6 -7). Injection was carried out in the early light phase following an overnight fast. *, p Ͻ 0.01 versus saline-injected controls; †, p Ͻ 0.05 versus leptin-alone groups. D and E, effects of NF-B inhibitor, pyrrolidine dithiocarbamate (100 M) on leptin-stimulated POMC promoter activity and POMC mRNA expression in AtT-20 cells. *, p Ͻ 0.005 versus untreated control; †, p Ͻ 0.05 versus leptin alone groups. F and G, intrahypothalamic administration of leptin caused anorexia and weight loss in Ikk␤ F/F mice. These effects were significantly attenuated in Ikk␤ ⌬POMC mice (n ϭ 5-6). *, p Ͻ 0.05; **, p Ͻ 0.01 versus saline-injected controls; †, p Ͻ 0.05 versus leptin-injected Ikk␤ F/F mice. Each bar represents mean Ϯ S.E. H, diagram summarizing our data. Hypothalamic NF-B is activated by infectious agents, LPS and Tat, and leptin to stimulate hypothalamic cytokine and POMC production, thereby contributing to anorexia and weight loss.
On the other hand, we have found that hypothalamic NF-B is activated by leptin, a critical anorexigenic hormone. Inhibition of hypothalamic NF-B blocks the anorexigenic and weight-reducing effects of leptin, suggesting an important role for hypothalamic NF-B in normal feeding regulation.
Consistently, Zhang et al. demonstrated that IKK␤ and NF-B are abundantly expressed in hypothalamic neurons (37). However, in contrast to our data, they showed that overnutrition induced activation of hypothalamic NF-B through elevated endoplasmic reticulum stress (37). Furthermore, hypothalamic NF-B activation interrupted leptin and insulin signaling by increasing the suppressor of cytokine signaling-3. Finally, depletion of IKK␤ in mediobasal hypothalamus and AGRP neurons had a protective effect against diet-induced obesity. From these data, they concluded that hypothalamic NF-B activation in obese state causes hypothalamic resistance to leptin and insulin.
Taking these data and our data together, hypothalamic NF-B plays a pivotal role in controlling feeding behaviors under the two opposite metabolic situations, undernutrition and overnutrition. Leptin-induced activation of NF-B acts as a downstream mediator of leptin in feeding regulation. On the other hand, NF-B activation may induce suppressor of cytokine signaling-3 expression, which would constitute a negative feedback pathway of leptin signaling, as is the case for the signal transduction-activated transcript-3 signaling pathway (38). It is also possible that NF-B activation in POMC neurons may have a differential role from that in AGRP neurons. Indeed, NF-B inhibition in POMC neurons did not show a protective effect against diet-induced obesity (supplemental Fig. S9).
The central melanocortin receptor activities are determined by the relative amounts of melanocortins and endogenous antagonist, AGRP at the receptors (25). Thus activation of melanocortin receptors also results from the decrease levels of AGRP. Indeed, hypothalamic AGRP mRNA expression showed a tendency of decrease following LPS and Tat administration ( Fig. 2A). Furthermore, treatment of LPS, Tat, and pro-inflammatory cytokines suppressed AGRP promoter activity in SH-SY5Y cells (supplemental Fig. S10). Thus NF-B-mediated suppression of AGRP may also contribute to infection-associated anorexia and weight loss, as suggested previously (39).
Chronic activation of hypothalamic NF-B and the melanocortin system during illness may have a detrimental effect on the host by causing a negative energy balance. However, cumulating evidence indicates that melanocortins have antipyretic, anti-inflammatory, and neuroprotective effects on both the central and peripheral nervous systems (26). In line with these notions, POMC-specific IKK␤ knock-out mice showed decreased activation of the hypothalamo-pituitary-adrenal axis and enhanced febrile reaction in response to LPS. Therefore, NF-B-mediated POMC activation may have both positive and negative effects on the hosts during infection.
Although we focused on hypothalamic events during infection, illness-associated anorexia and cachexia may be also mediated by peripheral or non-hypothalamic mechanisms. Indeed, LPS-and Tat-induced reduction in food intake and body weight was partially blocked by hypothalamic administration of NF-B and IKK inhibitors or in POMC-specific NF-B knock-out mice, when LPS and Tat were injected peripherally (data not shown). On the other hand, we did not expand our study to include the cancer-anorexia model. However, cancer-associated anorexia may be through a similar molecular mechanism, because hypothalamic IL-1␤ expression was increased in the hypothalamus from tumorbearing rats (40). Furthermore, blockade of hypothalamic melanocortins prevented IL-1␤or cancer-induced anorexia (41)(42)(43), indicating that hypothalamic NF-B and melanocortin may be a general molecular mediator causing anorexia and weight loss during chronic illness state.
In conclusion, we show that NF-B activation in the hypothalamic melanocortin system is essential for anorexia and weight loss induced by infectious agents and leptin. Therefore, chemicals having NF-B inhibitory activity or MC4R antagonistic activity may have a therapeutic potential on anorexia and cachexia.