Interleukin-7, a new cytokine targeting the mouse hypothalamic arcuate nucleus: role in body weight and food intake regulation

Abstract

Body weight is controlled through peripheral (white adipose tissue) and central (mainly hypothalamus) mechanisms. We have recently obtained evidence that overexpression of interleukin (IL)-7, a critical cytokine involved in lymphopoiesis, can protect against the development of diet-induced obesity in mice. Here we assessed whether IL-7 mediated its effects by modulating hypothalamic function. Acute subcutaneous injection of IL-7 prevented monosodium glutamate-induced obesity, this being correlated with partial protection against cell death in the hypothalamic arcuate nucleus (ARC). Moreover, we showed that IL-7 activated hypothalamic areas involved in regulation of feeding behavior, as indicated by induction of the activation marker c-Fos in neural cells located in the ventromedial part of the ARC and by inhibition of food intake after fasting. Both chains of the IL-7 receptor (IL-7Ralpha and gamma(c)) were expressed in the ARC and IL-7 injection induced STAT-3 phosphorylation in this area. Finally, we established that IL-7 modulated the expression of neuropeptides that tune food intake, with a stimulatory effect on the expression of pro-opiomelanocortin and an inhibitory effect on agouti-related peptide expression in accordance with IL-7 promoting anorectic effects. These results suggest that the immunomodulatory cytokine IL-7 plays an important and unappreciated role in hypothalamic body weight regulation.

Figure 1. IL-7 protects from MSG-induced body weight gain, increased fat mass, glucose- and insulin-resistance, and arcuate nucleus lesioning.

(A) Body weight evolution: Mice were injected with PBS (P) from post-natal day 5 (P5) to P12 (group P-P, n = 4;Δ), with monosodium glutamate (MSG) from P5 to P11 then PBS at P12 (group M-P, n = 4;○), with PBS from P5 to P11 then IL-7 at P12 (group P-7, n = 6;▴) or with MSG from P5 to P11 then IL-7 at P12 (group M-7, n = 6;•). Body weights were measured monthly during 6 months. Results are expressed as mean ± SEM. * p<0.05. (B) Visualization of the fat mass of one representative mice per group using Dual Energy-X-ray Absorptiometry. (C) Glucose tolerance test: At the age of 5 months, P-P (Δ), P-7 (▴), M-P (○) and M-7 (•) mice were fasted 18 h before i.p. glucose injection. Glycaemia was measured for each individual animal, from t = 0 to t = 330 min after glucose administration. Results are presented as mean ± SEM. Statistical analysis was performed with a 2-way ANOVA. (D) Insulin tolerance test: At the age of 5 months, P-P (Δ), P-7 (▴), M-P (○) and M-7 (•) mice were fasted 4 h before i.p. insulin injection. Glycaemia was measured from t = 0 to t = 105 min after insulin injection. Results are presented as mean ± SEM. Statistical analysis was performed with a 2-way ANOVA. * p<0.05. (E) Representative microphotographs of frontal hypothalamic sections stained with cresyl violet staining. Brains were harvested from 2-month-old P-P (upper left), P-7 (down left), M-P (upper right) and M-7 (down right) animals. Arrows show the partial cell survival in the M-7-treated group in comparison with the M-P group. Scale bar represents 100 µm. ARC: arcuate nucleus, ME: median eminence, 3^rd V: third ventricle, VMH: ventromedial hypothalamic nucleus.

Figure 2. IL-7 improves neural cell-survival.

(A) Bromodeoxyuridine (BrdU) treatment experimental procedure. (B) BrdU positive cells in the ARC of PBS- (□) or IL-7- (▪) treated mice. Animals were sacrificed one day after BrdU treatment (post-natal day 8 (P8)) for neural cell proliferation analysis (n = 5 per group), or 29 days after BrdU treatment (P36) for neural cell survival analysis (n = 5 per group). Data were represented as mean ± SEM of BrdU-positive cells within the ARC per animal and per slice. *p<0.05.

Figure 3. IL-7 activates hypothalamic cells and IL-7 receptor is expressed on hypothalamic cells.

(A) Representative microphotographs of Fos expression induced by peripheral IL-7 injection in C57BL/6 mice. Fos-immunoreactive cells in arcuate nucleus (ARC) in PBS-treated and IL-7-treated mice. Arrows indicate Fos-imunoreactive cells. 3^rd V: third ventricule; VMH: ventromedial hypothalamic nucleus. Scale bars represent 100 µm. (B) mRNA expression of IL-7Rα and γ_c chains in the whole hypothalamus of 2-month-old C57BL/6 mice (n = 3). Lanes (1, 2, 3) represent one animal. The - lane represents the negative control i.e. without retro-transcription. (C) Representative microphotographs of frontal hypothalamic frozen sections from 2-month-old mice showing fluorescent immunostaining of IL-7Rα and γ_c chains in the arcuate nucleus. Frozen section from IL-7Rα KO were used as control for the IL-7Rα specific staining. ARC: arcuate nucleus, ME: median eminence, 3^rd V: third ventricule; VMH: ventromedial hypothalamic nucleus. (D) Mean number of phosphorylated (p)-STAT5 and p-STAT3 immunoreactive (IR) cells in the hypothalamic arcuate nucleus of C57BL/6 mice injected with recombinant IL-7 (▪; n = 5) or with PBS (□; n = 5). Results are expressed as mean of immunoreactive cells ±SEM per animal and per slice. *p<0.05. (E) Representative microphotographs of p-STAT5 and p-STAT3-IR cells in the ARC in PBS-treated and IL-7-treated mice. Scale bar represent 100 µm. ARC: arcuate hypothalamic nucleus; ME: median eminence; 3rd: third ventricle.

Figure 4. IL-7 modulates hypothalamic neuropeptide mRNA gene expression.

PBS ( = 5, □) or IL-7 (n = 5, ▪) was i.p. injected in 2-month-old C57BL/6 mice. The expression of hypothalamic neuropeptides was assessed by real-time PCR, in fed (A) or in re-fed (B) condition. The effects of IL-7 treatment were evaluated by calculating the relative expression levels as follows: 2^ΔCt (ΔCt = mean Ct genes of interest - mean Ct GAPDH), using the raw cycle-threshold (Ct) values. *p<0.05, *** p<0.001. POMC: pro-opiomelanocortin, CART: cocaine-amphetamine related peptide, NP-Y: neuropeptide-Y, AgRP: Agouti-related peptide.

Figure 5. IL-7 regulates food intake after fasting.

(A) Food intake during basal conditions in IL-7- (▪) or PBS- (□) treated C57BL/6 mice (n = 7 per group). Results are expressed as the mean ± SEM of cumulative food intake in g/g of body weight. (B) Animals were overnight-fasted, treated with IL-7 (▪) or PBS (□) before getting free access to food (n = 5 per group). Food intake was measured during the 5 h following re-access to food and was expressed as mean ± SEM of cumulative food intake in g/g of body weight. *p<0.05. (C) Prior to the taste aversion assay, water was removed from animal cages overnight. Taste solution of 5% sucrose was offered for 30 min and mice were either s.c. injected with 0.3 µg of IL-7 (n = 5,•) or PBS as control (n = 4,○), or i.p. injected with 6 mEq of LiCl (n = 5,▪) or NaCl as control (n = 4,□). After injection, animals got free access to water containing 0.5% of sucrose. Sucrose cumulative consumption was individually measured 1 h, 3 h, 6 h and 24 h after injection. Results are expressed as mean of sucrose consumption in ml/body weight in g ± SEM. *p<0.05.