Removing melatonin receptor type 1 signaling leads to selective leptin resistance in the arcuate nucleus

Abstract

Recent studies have highlighted the involvement of melatonin in the regulation of energy homeostasis. In this study, we report that mice lacking melatonin receptor 1 (MT₁ KO) gained more weight, had a higher cumulative food intake, and were more hyperphagic after fasting compared to controls (WT). In response to a leptin injection, MT₁ KO mice showed a diminished reduction in body weight and food intake. To evaluate hypothalamic leptin signaling, we tested leptin-induced phosphorylation of the signal transducer and activator of transcription 3 (STAT3). Leptin failed to induce STAT3 phosphorylation in MT₁ KO mice beyond levels observed in mice injected with phosphate-buffered saline (PBS). Furthermore, STAT3 phosphorylation within the arcuate nucleus (ARH) was decreased in MT₁ KO mice. Leptin receptor mRNA levels in the hypothalamus of MT₁ KO were significantly reduced (about 50%) compared to WT. This study shows that: (a) MT₁ deficiency causes weight gain and increased food intake; (b) a lack of MT₁ signaling induces leptin resistance; (c) leptin resistance is ARH region-specific; and (d) leptin resistance is likely due to down-regulation of the leptin receptor. Our data demonstrate that MT₁ signaling is an important modulator of leptin signaling.

Keywords: leptin receptor; leptin resistance; melatonin; melatonin receptor 1; metabolism; obesity.

Figure 1.. MT₁KO gain more body weight gain and have higher cumulative food intake.

(A) Body weight gain over the course of 5 weeks of monitoring (mice were 12 weeks old at the start of the experiment). Two-way ANOVA followed by the Sidak post-hoc test. (B) Final body weight after 5 weeks of monitoring. Unpaired t-test. (C) Daily food intake. One-way ANOVA followed by the Sidak post-hoc test. (D) Cumulative food intake over the course of 20 days of monitoring. Two-way ANOVA followed by the Sidak post-hoc test. Data are shown as mean ± SEM; n = 8.

Figure 2.. No differences in adiposity and leptin levels in WT and MT₁KO mice.

(A) Percentage of Adipose Tissue Pad: subcutaneous (SC), retroperitoneal (RP) and periepydidimal (PE). Fat mass was normalized to body weight. Two-way ANOVA followed by the Sidak post-hoc test. (B) Leptin serum level. Unpaired t-test. Data are shown as mean ± SEM; n = 8.

Figure 3.. Removal of MT1 increases hyperphagia induced by fasting.

(A) Percentage of body weight loss over the course of a 12-h fasting test. (B) Food intake during the re-feeding period. Two-way repeated measures ANOVA followed by the Sidak post-hoc test. Data are shown as mean ± SEM; n = 8.

Figure 4.. Removal of MT₁ signaling influences acute leptin responsiveness test.

(A) Percentage of body weight gain 24 h after PBS or recombinant leptin injection (1.5 μg/g). (B) Food intake 24 h after PBS or recombinant leptin injection (1.5 μg/g). (C) Percentage of body weight gain 24 h after PBS or recombinant leptin injection (3 μg/g). (D) Food intake 24 h after PBS or recombinant leptin injection (3 μg/g). Two-way repeated measures ANOVA followed by the Sidak post-hoc test. n = 8. (E) Hypothalamic phosphorylation of STAT3 90 minutes after injection with PBS or leptin (1.5 μg/g), measured by Western Blot. Data were normalized by STAT3 and GAPDH amount. (F) Representative immunoblotting of pSTAT3, STAT3 and GAPDH protein content. Two-way ANOVA followed by the Sidak post-hoc test, n = 4. Data are shown as mean ± SEM.

Figure 5.. Site-specific difference in leptin-induced phosphorylation of STAT3 in the hypothalamus of WT and MT₁KO mice.

(A) Number of pSTAT3-immunoreactive cells in the ARH, DMH, VMH and PMv; *ARH WT vs. MT₁KO group p < 0.05. (B–I) Photomicrographs of coronal sections illustrating leptin-induced pSTAT3-immunoreactivity (pSTAT3-ir) in WT (B, D, F and H) and MT₁KO (C, E, G and I) mice in the ARH (B, C), DMH (D, E), VMH (F, G) and PMv (H, I). Scale bar = 100 μm. Abbreviations: 3v, third ventricle. Unpaired t-test. Data are shown as mean ± SEM; n =3.

Figure 6.. Removal of MT₁ signaling affects the transcription in the hypothalamus of genes controlling feeding behavior.

(A) Hypothalamic SOCS3 90 minutes after injection with PBS or leptin (1.5 μg/g), measured by Western Blot. Data were normalized by GAPDH amount. The upper insert shows representative immunoblotting of SOCS3 and GAPDH protein content. (B) mRNA levels of Socs3, Interaction, ZT and group factors p > 0.05; (C) mRNA levels of ObRb, Interaction p > 0.05, ZT factor p < 0.05 and group factor p < 0.001; (D) mRNA levels of Npy, Interaction p < 0.05, ZT and group factors p > 0.05; (E) mRNA levels of Agrp, Interaction p < 0.05, ZT and group factors p > 0.05 and (F) mRNA levels of Pomc, Interaction p < 0.05, ZT and group factors p > 0.05 in WT and MT₁KO mice over 24 h. Data were normalized by the geometric mean of Rpl and Ppia. Two-way ANOVA followed by the Sidak post-hoc test. Data are shown as mean ± SEM; n = 4.