NF-kappaB activation in hypothalamic pro-opiomelanocortin neurons is essential in illness- and leptin-induced anorexia

Abstract

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-kappaB (NF-kappaB), 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-kappaB. In vitro, NF-kappaB 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-kappaB 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-kappaB and melanocortin. Furthermore, disruption of I kappaB kinase-beta, an upstream kinase of NF-kappaB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-kappaB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-kappaB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-kappaB also serves as a downstream signaling pathway of leptin.

FIGURE 1.

Administration of LPS and Tat causes NF-κB activation and pro-inflammatory cytokine production in the hypothalamus. A, hypothalamic NF-κB activation following intraperitoneal administration of LPS. Mediobasal hypothalami were collected 1 h after intraperitoneal injection in the early light phase. B, LPS-induced NF-κB binding to the oligonucleotides was inhibited and supershifted in the presence of p50- and p65-specific antibodies, suggesting that canonical NF-κB pathway is activated. “C” represents saline-injected controls. C, triple immunohistochemistry with p50, neuron marker (MAP2), and nuclear marker (4′,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 = 3). p < 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-κB inhibitor (κB inh.) (n = 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 ± S.E. *, p < 0.005 versus saline-injected control (C); †, p < 0.05 versus Tat alone group.

FIGURE 2.

NF-κB regulates POMC transcriptional activity. A, hypothalamic neuropeptide mRNA expression after intraperitoneal administration of LPS and Tat_1–72 (n = 5–6). *, p < 0.05 versus saline-injected control. B, potential κB binding sites in the POMC genes. NF-κB consensus sequences are underlined. The shaded region indicates conserved sequences in promoter variants of human, mouse, and rat POMC genes. C, the promoter activity of the POMC region, including κB-2, was increased by expression of p50 and/or p65 in AtT-20 cells (n = 3). *, p < 0.01 versus mock vector-expressing controls; ^†, p < 0.05 versus p50 (50 ng) alone group. D, EMSA using oligonucleotides, including κB-1 and κB-2, with or without p50 antibody. Hypothalami were collected 1 h after intraperitoneal injection of saline (control) or LPS (200 μg/kg). E, nucleotide substitution within κB-2 blocked p50-induced increase in POMC promoter activity. *, p < 0.05 versus mock vector-expressing control. F, a chromatin immunoprecipitation assay revealed that LPS enhanced endogenous NF-κB binding to κB-2 sites in SH-SY5Y neuron cells. G, treatment with LPS, Tat_1–72, IL-1β, and tumor necrosis factor-α increased POMC promoter activities in AtT-20 cells. Cotreatment of the NF-κB inhibitor, Bay 11-7085 suppressed these effects (n = 3). *, p < 0.005 versus LPS-, Tat-, and cytokines-untreated controls; †, p < 0.005 versus Bay-untreated groups. H, depletion of p50 using murine p50-specific small interfering RNA completely inhibited LPS (10 ng/ml)- and Tat_1–72 (1 nm)-induced increase in POMC promoter activity in AtT-20 cells (n = 3). *, p < 0.05 versus LPS- and Tat-untreated and control small interfering RNA-treated controls; NS, not significant. Each bar represents mean ± S.E.

FIGURE 3.

Inhibition of hypothalamic NF-κB and POMC prevents LPS- and Tat-induced anorexia and weight loss. A and B, prior intrahypothalamic administration of the NF-κB inhibitor Bay 11-7085 inhibited LPS- and Tat_1–72-induced decrease in food intake and body weight (n = 6–7). C and D, intrahypothalamic administration of IKK inhibitory peptide prevented LPS-induced anorexia and weight loss (n = 5–6). E and F, prior ICV administration of the melanocortin antagonist, AGRP, blocked the effects of LPS and Tat_1–72 on food intake and body weight (n = 5–6). *, p < 0.05, **, p < 0.01 versus vehicle-injected controls, †, p < 0.05; ††, p < 0.01 versus Tat or LPS alone groups. Each bar represents mean ± S.E.

FIGURE 4.

NF-κB inactivation in POMC neurons blocks LPS- and Tat-induced anorexia. A, double immunofluorescence staining using antibodies against IKKβ and αMSH in Ikkβ^F/F mice and Ikkβ^ΔPOMC mice. B and C, basal hypothalamic NF-κB activity and POMC mRNA expression in Ikkβ^ΔPOMC and Ikkβ^F/F mice (n = 4–5). The mediobasal hypothalami were collected following a 5-h fast. D, LPS-induced nuclear translocation of p50 in POMC neurons was reduced in Ikkβ^ΔPOMC mice. A representative figure and the counts of intranuclear p50-immunoreactive spots in ARC POMC-producing neurons are presented. *, p < 0.01 versus saline-injected controls. NS: not significant. E–H, intrahypothalamic administration of LPS and Tat_1–72 caused anorexia and weight loss in Ikkβ^F/F mice. In Ikkβ^ΔPOMC mice, these effects were significantly attenuated (n = 5–6), which was recovered by coadministration of α-MSH. *, p < 0.05; **, p < 0.01 versus saline-injected Ikkβ^F/F and Ikkβ^ΔPOMC mice; †, p < 0.05 versus LPS- or Tat_1–72-injected Ikkβ^F/F mice. I, the LPS-induced increase in plasma cortisol concentrations was reduced in Ikkβ ^ΔPOMC mice, compared with Ikkβ ^F/F mice (n = 5–6). *, p < 0.05; **, p < 0.005 versus saline-injected groups; †, p < 0.05 versus LPS-injected Ikkβ^F/F mice. J, LPS-induced elevation in body temperature was greater in Ikkβ ^ΔPOMC mice than in Ikkβ^F/F mice (n = 5–6). *, p < 0.05; **, p < 0.01 versus saline-injected groups; †, p < 0.05 versus LPS-injected Ikkβ^F/F mice. Each bar represents mean ± S.E.

FIGURE 5.

Leptin-induced hypothalamic NF-κB activation mediates the effects of leptin on food intake, body weight, and POMC transcription. A, hypothalamic NF-κB activation following intraperitoneal administration of leptin (3 mg/kg) and LPS (200 μg/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 = 6–7). Injection was carried out in the early light phase following an overnight fast. *, p < 0.01 versus saline-injected controls; †, p < 0.05 versus leptin-alone groups. D and E, effects of NF-κB inhibitor, pyrrolidine dithiocarbamate (100 μm) on leptin-stimulated POMC promoter activity and POMC mRNA expression in AtT-20 cells. *, p < 0.005 versus untreated control; †, p < 0.05 versus leptin alone groups. F and G, intrahypothalamic administration of leptin caused anorexia and weight loss in Ikkβ^F/F mice. These effects were significantly attenuated in Ikkβ^ΔPOMC mice (n = 5–6). *, p < 0.05; **, p < 0.01 versus saline-injected controls; †, p < 0.05 versus leptin-injected Ikkβ^F/F mice. Each bar represents mean ± S.E. H, diagram summarizing our data. Hypothalamic NF-κB is activated by infectious agents, LPS and Tat, and leptin to stimulate hypothalamic cytokine and POMC production, thereby contributing to anorexia and weight loss.