Nuclear factor κB (NF-κB) suppresses food intake and energy expenditure in mice by directly activating the Pomc promoter
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While chronic low-grade inflammation is associated with obesity, acute inflammation reduces food intake and leads to negative energy balance. Although both types of inflammation activate nuclear factor κB (NF-κB) signalling, it remains unclear how NF-κB activation results in opposite physiological responses in the two types of inflammation. The goal of this study was to address this question, and to understand the link between inflammation and leptin signalling.
We studied the ability of NF-κB to modulate Pomc transcription, and how it impinges on signal transducer and activator of transcription 3 (STAT3)-mediated leptin signalling by using a combination of animal models, biochemical assays and molecular biology.
We report that suppression of food intake and physical movement with acute inflammation is not dependent on STAT3 activation in pro-opiomelanocortin (POMC) neurons. Under these conditions, activated NF-κB independently leads to increased Pomc transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) experiments reveal that NF-κB v-rel reticuloendotheliosis viral oncogene homologue A (avian) (RELA [also known as p65]) binds to the Pomc promoter region between −138 and −88 bp, which also harbours the trans-acting transcription factor 1 (SP1) binding site. We found significant changes in the methylation pattern at this region and reduced Pomc activation under chronic inflammation induced by a high-fat diet. Furthermore, RELA is unable to bind and activate transcription when the Pomc promoter is methylated. Finally, RELA binds to STAT3 and inhibits STAT3-mediated promoter activity, suggesting that RELA, possibly together with forkhead box-containing protein 1 (FOXO1), may prevent STAT3-mediated leptin activation of the Pomc promoter.
Our study provides a mechanism for the involvement of RELA in the divergent regulation of energy homeostasis in acute and chronic inflammation.
KeywordsAppetite Energy homeostasis FOXO1 Inflammation NF-κB Obesity STAT3
Agency for Science, Technology and Research
Electrophoretic mobility shift assay
Forkhead box-containing protein 1
Nuclear factor κB
POMC-neuron-specific deletion of Stat3 (Pomc/Stat3F/F)
Protein-tyrosine kinase 1B
Ras-related protein 1
V-rel reticuloendotheliosis viral oncogene homologue A (avian)
Respiratory exchange ratio
Suppressor of cytokine signalling 3
Trans-acting transcription factor 1
Signal transducer and activator of transcription 3
The maintenance of energy homeostasis is regulated by neurohormonal signals. Leptin, a key hormone involved in this process, is released from adipocytes . Through a saturated transport mechanism, circulating leptin enters the brain and elicits specific downstream signalling pathways to inhibit food intake and energy expenditure by acting on at least the following neurons in the hypothalamus: the orexigenic neuropeptide Y (NPY) and the anorexigenic pro-opiomelanocortin (POMC) neurons [1, 2]. In the case of POMC neurons, leptin binds to the long-form leptin receptor , leading to phosphorylation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3). Activated STAT3 then binds to the trans-acting transcription factor 1 (SP1)–Pomc promoter complex to activate Pomc transcription to suppress appetite [2, 4].
However, the leptin signalling can be impaired in pathological conditions, such as in obesity models, resulting in a phenomenon known as leptin resistance . A number of models have been proposed to account for leptin resistance, for example, suppression of STAT3 phosphorylation and thus its downstream actions by suppressor of cytokine signalling 3 (SOCS3) [6, 7] and protein-tyrosine phosphatase 1B (PTP1B) [8, 9]. Previously, we have identified that forkhead box-containing protein 1 (FOXO1) may act at a step downstream of STAT3 phosphorylation to prevent activated STAT3 from interacting with the SP1–Pomc promoter complex, thereby repressing transcriptional activation . FOXO1 may also inhibit phosphorylated STAT3 (pSTAT3) action directly at the promoter level [11, 12].
Other non-physiological conditions, such as inflammation, can also have direct and profound effects on food intake and energy expenditure . Acute inflammation such as that caused by bacterial lipopolysaccharide (LPS) activates TNF-α, IL-1β, and IL-6 in the hypothalamic–pituitary cytokine network and subsequently induces Pomc expression, leading to reduced food intake and increased energy expenditure . In contrast, chronic low-grade inflammation is often associated with unsuppressed appetite and increased obesity, along with other metabolic disorders, such as insulin resistance and beta cell dysfunction, although inflammatory cytokines are also elevated under this condition.
Inflammatory cytokines bind to cell surface toll-like receptors (TLRs) to activate the IκB kinase (IKK) kinase complex, which consists of the catalytic subunits IKKα and IKKβ, and the regulatory subunits IKKγ (also known as NF-κB essential modulator [NEMO]), Ras-related protein 1 (RAP1)  and ELKS (also known as ELKS/RAB6-interacting/CAST family member 1 [ERC1]) . IKKβ plays a key role in metabolic regulation. For example, mice with brain- and hypothalamus-specific deletion of Ikkβ (also known as Ikbkb) show decreased food intake, and are protected against diet-induced obesity, insulin resistance and glucose intolerance [17, 18, 19]. The IKK kinase complex is crucial for activation of nuclear factor κB (NF-κB) to drive the expression of a large number of target genes, which may intersect with other signalling cascades such as those controlled by leptin. The fact that both acute and chronic inflammation activate NF-κB, but result in opposite physiological responses, indicates that NF-κB does not simply antagonise leptin signalling.
To understand the role of NF-κB in energy regulation, and to tease out the link between inflammation and leptin signalling pathways, we studied Pomc transcriptional regulation by v-rel reticuloendotheliosis viral oncogene homologue A (avian) (RELA [also known as p65]), the most abundant subunit of NF-κB , and STAT3, a key regulator in leptin signalling. Here, we show that RELA activation in acute inflammatory conditions directly promotes Pomc transcription. The direct activation of Pomc promoter, however, is impaired under chronic inflammatory conditions, as RELA is unable to bind to methylated Pomc promoter, which occurs under chronic inflammation. At the same time, like FOXO1, RELA also attenuates leptin action by binding to STAT3. The combined effect under chronic inflammatory conditions is loss of Pomc regulation by either RELA or STAT3. These in vitro findings support our model of divergent regulation of Pomc transcription by NF-κB, which sheds new light on how inflammation may interfere with leptin signalling. Future in vivo studies are needed to validate this model.
All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Agency for Science, Technology and Research (A*STAR). The mice were supplied by the A*STAR animal facility in Singapore.
Oxymax/Comprehensive Lab Animal Monitoring System
Oxymax/Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH, USA) was used to measure and determine oxygen consumption (volume of O2 [VO2]), carbon dioxide production (volume of CO2 [VCO2]), activity, food intake, basal metabolic rate, respiratory exchange ratio (RER) and movements of individual mice [21, 22]. Paired mice were individually housed in chambers maintained at 24 ± 1°C, and given free access to chow and water. All the measurements were taken every 15 min for 5 days after the mice were acclimatised for 1 day. All data collected were averaged from 5 days’ monitoring.
Mouse tissue preparation, DNA extraction and bisulfite modification
We used male C57Bl/6 mice fed with a high-fat diet (HFD) containing 60% of energy from fat, or a low-fat diet (LFD) (Research Diets, New Brunswick, NJ, USA) for 20 weeks (n = 20 per group). Mouse brains were quickly removed and frozen using Mouse Brain Slicer Matrix (Zivic Instruments, Pittsburgh, PA, USA) on dry ice. Coronal sections, 500 μm containing the arcuate (Arc) region of the hypothalamus were collected by microdissection knife for genomic DNA isolation by DNeasy kit (Qiagen, Valencia, CA, USA), followed by bisulfate modification and purification using the EpiTect Bisulfite Kit (Qiagen).
Amplification of the Pomc promoter region, ligation, cloning and sequencing
The region between −269 and −99 bp in the Pomc promoter was amplified with the following primers (5′-TTG TTT AGT TTT AAG TGG AGA TTT AAT ATT-3′ and 5′-AAA ACT ATC CAA AAC TAA AAC ACC CT-3′) using bisulfite-modified DNA as templates. After purification with QIAquick Gel Extraction Kit (Qiagen), the PCR products were ligated into the TA vectors (pCT2.1-TOPO, Qiagen). Sequencing for the Pomc promoter region was performed on 15–20 positive clones per mouse.
Quantitative real-time PCR
The cDNAs were diluted ten times for PCR analysis in triplicates. A 10 μl volume of reaction mixture contained 5 μl SYBR Green, PCR Master Mix, 300 nmol/l specific target gene primers and 2 μl of cDNA. The reactions were performed on an ABI Prism 7500 sequence detector system. SYBR Green primers were designed by Primer Express software from Applied Biosystems (Carlsbad, CA, USA); the sequences are provided in electronic supplementary material (ESM) Table 1.
The Pomc promoter-luciferase construct (Pomc-Luc) was a generous gift from D. Accili (Columbia University, New York, NY, USA). The following plasmids were generated in our laboratories and have been described previously: pcDNA3.1-Rela-Myc, pXJ40-Flag-STAT3, pN3-Flag-Sp1 .
Cell culture and luciferase assay
Flp-In 293-OBRb (293-OBRb) cells  were cultured in Dulbecco’s Minimal Essential Medium (Invitrogen, Carlsbad, CA, USA) containing 10% (vol./vol.) fetal bovine serum in a 37°C incubator with 5% CO2. Transfection and cell treatment were done as previously described . Luciferase activity was measured from cell extracts on an Lmax II (Molecular Devices, Sunnyvale, CA, USA). The firefly luciferase activity was normalised to Renilla luciferase activity.
Cytosolic and nucleus extracts preparation
The cells were washed twice and collected in cold PBS. The cell suspension was centrifuged at 200 g for 5 min. The resulting pellet was then resuspended with hypotonic buffer containing 20 mmol/l HEPES, pH 7.9, 10 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l Na3VO4, 10% glycerol, 0.2% Nonidet P-40, 20 mmol/l NaF, 1 mmol/l dithiothreitol and ×1 complete protease inhibitor (Roche Applied Science, Indianapolis, IN, USA) and rocked at 4°C for 10 min. The mixture was then centrifuged at 20,000 g for 30 s, the supernatant fraction was collected as the cytosolic fraction, and the pellet was resuspended with high-salt buffer (20% glycerol, 420 mmol/l NaCl, 1 mmol/l Na3VO4, 1 mmol/l dithiothreitol and ×1 complete protease inhibitor in hypotonic buffer without Nonidet P-40). After 1 h rocking, the mixture was centrifuged at 20,000 g for 10 min at 4°C. The supernatant fraction was then collected as the nuclear extract.
293-OBRb cells were transfected with expression vectors pcDNA3.1-Rela-Myc (full length or deletion mutants) and pXJ40-Flag-Stat3 (full length or deletion mutants) by using Lipofectamine 2000 (Invitrogen) and harvested 48 h after transfection with whole-cell lysis buffer (20 mmol/l Hepes pH 7.9, 280 mmol/l KCl, 1 mmol/l EDTA, 0.1 mmol/l Na3VO4, 10% Glycerol, 0.5% NP40, 1 mmol/l DTT, 1 mmol/l PMSF, protease inhibitor cocktail). Cell lysate, 1,000 μg, was incubated with antibody against FLAG (Sigma-Aldrich, St Louis, MO, USA), MYC (Roche Applied Science) or control mouse IgG overnight, followed by immunoprecipitation with 1:1 protein A/G-sepharose beads (Invitrogen) for 1 h. The beads were washed four times with lysis buffer, mixed with ×2 SDS loading buffer, and the resulting samples were loaded and resolved using 10% SDS-PAGE.
Chromatin immunoprecipitation assays
Preparation of chromatin DNA from 293-OBRb cells expressing Flag-Rela or Flag-Sp1 and subsequent chromatin immunoprecipitation (ChIP) assays were done with a (ChIP) assay kit (Upstate Biotechnology, Waltham, MA, USA) according to the manufacturer's instructions. Rela-DNA or Sp1-DNA complexes were precipitated by anti-FLAG antibody (Sigma-Aldrich). Precipitated DNA was amplified by real-time PCR using primers flanking the potential RELA binding site (−138 to −88 bp) in the Pomc promoter (ESM Table 2). Rabbit IgG was used as the negative control.
Electrophoretic mobility shift assays
Nuclear, cytosolic or whole-cell protein extracts were collected using an NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific, Waltham, MA, USA). Normal, mutant and methylated probes (ESM Table 3) were annealed and labelled by biotin (LightShift Chemiluminescent EMSA Kit, Thermo Scientific). For 20 μl binding reaction, 2–3 μl of NE-PER nuclear extract was used. To each 20 μl binding reaction, 5 μl of ×5 loading buffer was added, and then 20 μl samples were loaded onto 6% polyacrylamide gel in ×0.5 Tris/borate/EDTA (TBE). After membrane transfer and crosslinking, supersensitive x-ray films (Kodak, Rochester, NY, USA) were exposed for 2–5 min to detect the biotin-labelled DNA by chemiluminescence.
SDS-PAGE and western blot
Electrophoresis of protein samples was done under denaturing reducing conditions using 10% polyacrylamide gels with SDS running buffer (Invitrogen). The blotted membranes were probed with antibodies against phosphorylated-STAT3 (pSTAT3 [Tyr705]), total STAT3, phosphorylated RELA (pRELA [Ser536]), RELA (Cell Signaling, Boston, MA, USA), FLAG (Sigma-Aldrich) and MYC (Roche Applied Science).
The data are presented as mean ± SEM. Comparisons of data were made using two-tailed Student’s t test for independent data. The significance limits are displayed as *p < 0.05, and **p < 0.01.
LPS-induced acute inflammation suppresses appetite and physical activity independent of STAT3-mediated Pomc activation
RELA directly activates Pomc transcription
RELA regulates Pomc transcription through direct interaction
Reduced Pomc expression levels in mice lacking Rela
To examine whether RELA regulates Pomc transcription in vivo, we studied mice lacking RELA on the background of Tnfr1 knockout. Mice with Rela deletion die in utero at embryonic day 14–15 because of massive liver destruction, with large areas of apoptosis [31, 32, 33], but can survive to about 40 days when tumour necrosis factor-receptor 1 (TNFR1)-mediated signalling is abolished by deleting Tnfr1. The Tnfr1−/−RelA−/− double-knockout (DKO) mice lack the development of lymph nodes, organised spleen structure, and T cell responses , and eventually die from acute infiltration of immature neutrophils in the liver [35, 36]. We performed real-time PCR to measure the mRNA levels in the hypothalamus of DKO mice and compared them with the Tnfr1−/− mice as control. Pomc expression levels decreased by ∼80% in the DKO mice, while expression of neuropeptides agouti-related peptide (AgRP) and NPY, and inflammatory cytokines IL-6 and TNF-α were not changed (ESM Fig. 1). These in vivo findings strongly suggest a specific role of RELA in the regulation of Pomc transcription in the hypothalamus.
Increased Pomc promoter methylation in mice fed an HFD
RELA inhibits leptin-stimulated POMC activity through STAT3 interaction
The first aim of this study was to understand how the two types of inflammatory response lead to opposite physiological effects in food intake and energy expenditure, even though both acute and chronic inflammation activates NF-κB signalling. Considering the critical importance of POMC neurons and the neuropeptides released from these neurons [2, 4, 43, 44], we focused on the regulation of Pomc transcription by various signalling pathways, in particular NF-κB signalling and leptin-induced STAT3 signalling.
At the onset of acute inflammation, such as viral infection, NF-κB RELA is released and translocates to the nucleus, where it binds directly to the Pomc promoter to induce Pomc transcription (Fig. 6b). Under this condition, the leptin level is very low. In the absence of leptin signalling, STAT3 is not phosphorylated, and remains in the cytosol, and thus does not contribute to the appetite suppression during acute inflammation.
The situation is more complex during chronic inflammation, which is often associated with dysregulated food intake and energy expenditure, i.e. leptin resistance. So why does activated NF-κB signalling in the hypothalamus induced by chronic inflammation fail to promote Pomc transcription? Numerous studies have established a clear link between epigenetic changes, including DNA methylation changes, and gene transcription activities [38, 45, 46, 47]. For example, the methylation status of the Pomc promoter was reported to change under unfavourable metabolic conditions [39, 40, 41, 42]. Our current study identified increased methylation at and near the RELA binding site within the Pomc promoter using tissues from the Arc region of HFD-fed mice (Fig. 4a). Moreover, RELA is unable to activate Pomc transcription when the promoter is hypermethylated, possibly because of an impaired interaction between RELA and the Pomc promoter (Fig. 4b, c). Therefore, even though RELA can be activated and translocated into the nucleus during chronic inflammation, increased DNA methylation prevents RELA from interacting with the Pomc promoter, resulting in the failure of NF-κB-signalling-induced Pomc transcription (Fig. 6c).
The second aim of the study was to investigate how chronic inflammation contributes to leptin resistance. To address this, we studied the crosstalk between NF-κB signalling and leptin signalling in the regulation of Pomc transcription. Two non-exclusive models may underlie leptin resistance depending on the steps relative to STAT3 phosphorylation. Leptin resistance may occur at a step upstream of STAT3 phosphorylation, leading to impaired or failed STAT3 activation. Molecules involved in this model include an SH2-domain-containing protein tyrosine phosphatase (SHP2) , SOSC3 [6, 7], and PTP1B [8, 9]. For example, the feedback inhibition of STAT3 by leptin–STAT3-pathway-induced upregulation of SOCS3 is a model for late-stage leptin resistance [48, 49]. Besides the classic activation of Socs3 transcription and expression by STAT3, NF-κB is another direct positive regulator of Socs3 transcription . Under chronic inflammation, the activated NF-κB signalling may thus lead to elevated SOCS3, which in turn suppresses STAT3 and the leptin signalling. Leptin resistance can also occur downstream of STAT3 phosphorylation. For example, FOXO1 inhibits pSTAT3 binding to the SP1–Pomc complex by sequestering STAT3 through direct interaction . The fact that RELA interacts with STAT3 [27, 28, 29, 30] and inhibits STAT3-mediated promoter activity further highlights the connection between inflammation and leptin signalling, and offers a novel direct link between the inflammatory pathway and leptin resistance. Similar to FOXO1 action in leptin resistance, RELA may also block leptin-induced POMC activity through direct interaction (Fig. 6c). Thus, our current work and the study by Zhang et al  provide a linkage between NF-κB and leptin signalling, and offer a model of how two parallel pathways acting both upstream and downstream of STAT3 activation may lead to leptin resistance in obesity. In our study, we used mice fed an HFD long term as a model for chronic inflammation. A recent study reported a hypothalamic inflammatory response in rodents within 1 to 3 days of HFD onset . Whether similar changes occur because of short-term HFD exposure remains to be determined.
In summary, in conditions of acute inflammation, RELA can directly activate Pomc transcription, whereas during chronic inflammation, increased promoter methylation leads to excess free RELA, which blocks leptin-induced signalling by interaction with STAT3. Our current in vitro findings support a novel mechanism for the divergent regulation of appetite control by NF-κB signalling under different inflammatory conditions, and for leptin resistance involving the crosstalk between NF-κB RELA and leptin signalling.
WH and GKR designed the research. XS, XW, QL, MS, EC, ETW performed the experiments. XS, XW, ZL, VT and WH analysed the data. XS, VT and WH wrote the manuscript. All authors were involved in the discussion and approved the manuscript.
Research in the laboratories of V. Tergaonkar and W. Han was supported by an intramural funding from the A*STAR Biomedical Research Council.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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