Fig 2: An RCN2 nonsense mutation contributes to NES.a Pedigree of the family with NES. The red arrow indicates the proband in this family. WT RCN2 genotypes are depicted as +/+, and heterozygous RCN2 genotypes are depicted as +/–. Numbers in red brackets indicate the BMIs of three affected obese patients. b Sanger sequencing chromatograms from II2, II3, III2 and one representative family member (II5). The c.469 C > T transition is indicated by the red arrow. c Protein homology of the neighboring region of the variant among selected species. The arrow shows the location of the p.Arg157Ter substitution. The red amino acid residue indicates the altered residue by the above variant found in this family. d–f Plasma Raptin levels of NES patients (II2, d; II3, e; III2, f) and their respective obese control (age-, sex- and BMI-matched, n = 6) during night phase. g, h The cumulative energy intake curves (g) and quantification (h) of II2 and his obese control (age-, sex- and BMI-matched, n = 6) during day and night phases. i, j The cumulative energy intake curves (i) and quantification (j) of II3 and her obese control (age-, sex- and BMI-matched, n = 6) during day and night phases. k, l The cumulative energy intake curves (k) and quantification (l) of III2 and his obese control (age-, sex- and BMI-matched, n = 6) during day and night phases. Data are shown as the mean ± SEM. See also Supplementary information, Fig. S13.

Fig 3: Raptin is a sleep-induced hypothalamic hormone in mice and humans.a Volcano plots of dysregulated factors ( ≥ 1.5-fold change) identified in the hypothalamus from sleep fragmentation mice (SF) compared to age-matched controls through proteomics and MS analysis (n = 3 per group). The red circle indicates Rcn2. b UMAP plot showing the clustering of cell types in the hypothalamus by analyzing multiple public scRNA-seq data from the GSE87544, GSE119960, GSE132355, GSE132730 and GSE148568 datasets. c Dot plots displaying Rcn2 expression in different cell types of mouse hypothalamus from scRNA-seq data. d Representative image of RCN2 (green) expression in the neurons (NeuN, violet, yellow arrows indicate the colocalization), microglia (IBA1, orange, white arrows) and astrocytes (GFAP, red, white arrows) of mouse brain slices. Scale bars, 50 μm. e, f Representative images (e) and quantification of RCN2 (green, f) expression in the PVN of mice at different time points (ZT0, ZT6, ZT12, ZT18). Scale bar, 50 μm (n = 5 per group). g Representative western blot of RCN2 expression in cell lysates and the concentrated culture medium of hypothalamic GT1-7 neurons transfected with the Flag-Rcn2 plasmid. The red triangle indicates Raptin (MW: 35–55 kD), and blue triangle indicates full-length RCN2 (MW: ~55 kD). h Schematic diagram illustrating the cleavage of RCN2 into Raptin. Raptin is spanning from 28 to 249 amino acids of full-length RCN2. i Plasma Raptin levels of 3-month male mice monitored at ZT0, ZT6, ZT12 and ZT18 (the purple area indicates the sleep phase: ZT0–ZT12). j Representative images of RCN2 (green) and NeuN (red) expression in the human hypothalamus section. Scale bar, 200 μm. k Plasma Raptin levels of humans monitored at ZT0, ZT6, ZT12 and ZT18 (purple area indicates sleep phase). Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA (f, i, k). See also Supplementary information, Figs. S1, S2.

Fig 4: The circadian rhythm of Raptin release is controlled by SCNAVP neurons.a Schematic diagram of the retrograde tracing system in SCN-innervating PVNRCN2. AAV-Rcn2-Cre-EGFP, AAV-DIO-H2B-T2A-TVA and AAV-DIO-RVG were mixed and then bilaterally injected into PVN. Three weeks later, rabies virus (RV)-EnvA-tdTomato (red) were bilaterally injected into PVN. b, c Representative images (b) and quantification (c) of colocalization with RV-tdTomato (red) by immunofluorescence staining of AVP+ neurons (top line, green), VIP+ neurons (middle line, green) and GRP+ neurons (bottom line, green) in SCN. Scale bars, 50 μm (n = 3 per group). d Schematic diagram of the anterograde tracing system in SCN-innervating PVN. The WT mice were bilaterally injected with scAAV2/1-hSyn-Cre into SCN and AAV-DIO-mCherry into PVN. e, f Representative images (e) and quantification (f) of colocalization with mCherry (red) by fluorescence ISH of Rcn2 mRNA (green) in PVN neurons. Scale bar, 50 μm (n = 3). g Schematic diagram of chemogenetic inhibition of SCNAVP neuron of Avp-Cre mice. AAV-DIO or AAV-DIO-hM4Di was injected into SCN of Avp-Cre mice. Mice were intraperitoneally injected with CNO at a dose of 2 mg/kg body weight. h, i Representative images (h) and quantification (i) of co-staining of c-Fos (red) and RCN2 (green) in PVN after CNO injection for 60 min to induce SCNAVP neuron inhibition in Avp-Cre mice. Scale bar, 50 μm (n = 4 per group). j Plasma Raptin levels of mice injected with AAV-DIO or AAV-DIO-hM4Di before and after intraperitoneal injection of CNO for 1 h (n = 4 per group). k Schematic diagram illustrating the optogenetic activation of SCNAVP neuron terminal in PVN, followed by the detection of PVNRCN2 neuron activity. AAV-DIO or AAV-DIO.ChR2 was bilaterally injected into SCN, whereas AAV-Rcn2-Cre-EGFP was injected into the PVN of Avp-Cre mice. The fiber was implanted into the PVN to stimulate SCNAVP neuronal terminal. l, m Representative traces (l) and action potential frequency (m) of PVNRCN2 neurons in brain slice before and during optogenetic activation of SCNAVP neurons (n = 3 ~ 4 per group). n The plasma Raptin levels before and after optogenetic activation of SCNAVP neurons in vivo (n = 3 ~ 4 per group). o Schematic diagram illustrating the experimental workflow for SCNAVP neuron activation and PVNRCN2 neuron inhibition. AAV-DIO-mCherry or AAV-DIO-hM4Di-mCherry with AAV-Rcn2-Cre-EGFP were injected into PVN for chemogenetic manipulation of PVNRCN2 neuron. CNO was intraperitoneally injected into mice at a dose of 2 mg/kg body weight. AAV-fDIO.ChR2 with AAV-Avp-Flp were injected into SCN for optogenetic activation of SCNAVP neurons. The fiber was implanted into the PVN to stimulate SCNAVP neuronal terminal in PVN. p, q The plasma Raptin levels in mice injected with AAV-DIO (p) or AAV-DIO-hM4Di (q) before and after CNO treatment (n = 4 per group). Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01 by a two-tailed paired Student’s t-test (j, m, n, p, q) or by a two-tailed unpaired Student’s t-test (i). See also Supplementary information, Figs. S3, S4.