gp130 signaling in proopiomelanocortin neurons mediates the acute anorectic response to centrally applied ciliary neurotrophic factor

Abstract

Ciliary neurotrophic factor (CNTF) exerts anorectic effects by overcoming leptin resistance via activation of hypothalamic neurons. However, the exact site of CNTF action in the hypothalamus has not yet been identified. Using Cre-loxP-mediated recombination in vivo, we have selectively ablated the common cytokine signaling chain gp130, which is required for functional CNTF signaling, in proopiomelanocortin (POMC)-expressing neurons. POMC-specific gp130 knockout mice exhibit unaltered numbers of POMC cells and normal energy homeostasis under standard and high fat diet. Endotoxin (LPS) and stress-induced anorexia and adrenocorticotropin regulation were unaffected in these animals. Strikingly, the anorectic effect of centrally administered CNTF was abolished in POMC-specific gp130 knockout mice. Correspondingly, in these animals, CNTF failed to activate STAT3 phosphorylation in POMC neurons and to induce c-Fos expression in the paraventricular nucleus. These data reveal POMC neurons as a critical site of CNTF action in mediating its anorectic effect.

Fig. 1.

Generation of POMC-specific knockout (gp130^ΔPOMC) mice. (a) PomcCre mice were mated with Z/EG reporter mice, and immunohistochemistry for enhanced GFP was performed in double-transgenic mice. (Scale bar: 100 μm.) (b) Western blot analysis of gp130 and pten (loading control) in brain, hypothalamus (HT), liver, and white adipose tissue (WAT) in control (CO) and gp130^ΔPOMC (KO) mice. (c) Double immunohistochemistry for p-STAT3 (green) and β-gal (red) in POMC neurons of control (gp130^+/+) and gp130^ΔPOMC (gp130^−/−) mice 30 min after i.v. stimulation with NaCl, leptin, or recombinant CNTF. (d) Quantification of p-STAT3-positive POMC cells after CNTF stimulation in control (CO) and gp130^ΔPOMC (KO) mice (mean ± SEM of three animals in each group) (P < 0.001). (e) Quantitative and spatial analysis of POMC neurons in control (open bars) and gp130^ΔPOMC (filled bars) mice (n = 3 of each genotype).

Fig. 2.

Unaltered stress response in gp130^ΔPOMC mice. (a) Immunohistochemistry for enhanced GFP performed in the pituitary of PomcCre, Z/EG mice. (b) Relative expression of Pomc mRNA in pituitaries of control (CO) and gp130^ΔPOMC (KO) mice (n = 7–8). (c) Plasma corticosterone concentrations: basal and 1 h after NaCl injection, restraint stress, or LPS treatment in control (open bars; n = 6–10) and gp130^ΔPOMC (filled bars; n = 7–12) mice. (d) IL-6 plasma concentrations: basal and 1 h after NaCl injection, restraint stress, or LPS treatment in control (open bars; n = 6) and gp130^ΔPOMC (filled bars; n = 7) mice.

Fig. 3.

Unaltered energy homeostasis in gp130^ΔPOMC mice. (a) Body-weight curve of control (open squares and open triangles) and gp130^ΔPOMC (filled squares and filled triangles) mice on standard diet (SD) (squares; n = 12–18) and high-fat diet (HFD) (triangles; n = 12–31). (b) Epigonadal fat pads were dissected and weighed. Data represent the mean ± SEM of 14–31 mice in each group. (c) Serum leptin levels of control (n = 10–16) and gp130^ΔPOMC (n = 10–16) mice on SD and HFD at the age of 24 weeks. (d) Daily food intake of control (n = 6) and gp130^ΔPOMC (n = 9) mice at the age of 11 weeks. (e) Basal metabolic rate (BMR) of control (CO) (n = 6) and gp130^ΔPOMC (KO) (n = 9) mice at the age of 11 weeks. (f) Relative expression of POMC and CART in control (open bars; n = 7–8) and gp130^ΔPOMC (filled bars; n = 6–7) mice. (g) Body length of control (n = 13) and gp130^ΔPOMC (n = 13) mice.

Fig. 4.

gp130^ΔPOMC mice are resistant to the acute anorectic effect of i.c.v. injected CNTF. i.c.v. injection of either 0.9% NaCl or 1 μg of recombinant rat CNTF into the lateral ventricle of 13- to 15-week-old mice. (a) Food intake of female control (open bars) and gp130^ΔPOMC (filled bars) mice, 0.9% NaCl-treated, 4 and 24 h after i.c.v. CNTF injection (mean ± SEM of 9–10 animals in each group) (P < 0.05, P < 0.01). (b) Body weight of female control (open bars) and gp130^ΔPOMC (filled bars) mice, 0.9% NaCl-treated, 4 and 24 h after CNTF injection (mean ± SEM of 9–10 animals in each group) (P < 0.05). (c) Food intake of male control (open bars) and gp130^ΔPOMC (filled bars) mice, 0.9% NaCl-treated, 6 h after i.c.v. CNTF injection (mean ± SEM of 6–16 animals in each group) (P < 0.01). (d) Body weight of male control (open bars) and gp130^ΔPOMC (filled bars) mice, 0.9% NaCl-treated, 6 h after i.c.v. CNTF injection (mean ± SEM of 6–16 animals in each group) (P < 0.05). (e) c-Fos expression in the PVN of control and gp130^ΔPOMC mice 6 h after i.c.v. injection of 0.9% NaCl or CNTF. (f) Quantification of c-Fos-positive cells in the PVN of control and gp130^ΔPOMC mice 6 h after i.c.v. injection of 0.9% NaCl or CNTF.