Journal: Nature Metabolism

Article Title: Feeding-regulated glycogen metabolism drives rhythmic liver protein secretion

doi: 10.1038/s42255-026-01453-8

Figure Lengend Snippet: a , Experimental design (created with BioRender.com ). WT mice were maintained under a 12 h light–dark cycle and exposed to NR feeding. Liver tissue was collected every 3 h over two consecutive days ( n = 16 (8 timepoints × 2 biological replicates)). b , Heatmap representation of the rhythmic microsomal proteome (adjusted P < 0.1) analysed across two independent days (day 1 and day 2). Data are row-standardized. c , Number of rhythmic microsomal liver proteins as a function of minimal amplitude. The colour code represents different false discovery rate (FDR) values. d , Phase distribution of rhythmic microsomal liver proteins. The colour code represents different FDR values. e , Scatterplot showing the relationship between acrophase and amplitude of rhythmic microsomal proteins. f , Examples of two rhythmic microsomal proteins confirmed by western blot: STX4 and ARFGAP1 (top). Quantification of western blot analysis (bottom). LC, loading control. MS: n = 16, 8 timepoints × 2 biological replicates; WB: n = 23, 8 timepoints × 3 biological replicates, except ZT21 (two biological replicates). g , Gene set enrichment analysis of rhythmic proteins in the secretory pathway. Adjusted P = 0.05, indicated by a dashed line. h , Phase distribution of microsomal rhythmic proteins associated with the ER and GA (statistical difference in phase evaluated by Kolmogorov–Smirnov test, P = 2.2 × 10⁻¹⁶). i , Scatterplot showing the relationship between acrophase and amplitude of rhythmic ER-associated (left) and GA-associated (right) proteins. j , Representative images (left) and quantitative analysis (right) of electron microscopy imaging of mouse livers at one timepoint during the day (ZT4) and night (ZT16) under NR feeding ( n = 4 biological replicates with GA ZT16 ( n = 3). ER and GA are highlighted in orange and blue, respectively. N, nucleus; mt, mitochondria. Each colour in the graphs represents an independent biological replicate. Several independent liver areas per biological replicate were analysed. k , Heatmap representation of rhythmic proteins involved in protein glycosylation, categorized by function. Data are row-standardized. l , Scatterplot showing the relationship between acrophase and amplitude of N -glycans on the indicated protein. Non-complex (oligomannose) and complex glycosylation modifications. m , Phase difference between rhythmic oligomannose (top) and complex (bottom) N -glycans in mouse liver (two-tailed Kolmogorov–Smirnov test for the difference in phase, P = 0.011). n , Temporal profiles of the microsomal proteins and N -glycans at indicated sites of RAB2 and H2-K1. Temporal profiles of microsomal proteins, oligomannose and complex N -glycans at indicated positions ( n = 16 (8 timepoints × 2 biological replicates)). Data are displayed as means; error bars, s.e.m. Tukey boxplots show the median and interquartile range, whiskers extend to the most extreme values within 1.5× the interquartile range and outliers are shown as individual points. A detailed description of the statistical analysis is available in Source Data Fig. . See also Extended Data Fig. and Supplementary Tables and for related data.

Article Snippet: Primary antibodies were used at the following dilutions: 1:1,000 for ATF4 (Cell Signaling Technologies, 11815), ARFGAP1 (Cell Signaling Technologies, 14608), Phospho-RPS6 (Cell Signaling Technologies, 2211), Total-RPS6 (Cell Signaling Technologies, 2217), GABARAPL1 (Genetex, GTX132664) and ConA Lectin (Vector Laboratories, B-1005) and 1:2,000 for STX4 (ProteinTech, 14988-1-AP).

Techniques: Western Blot, Control, Electron Microscopy, Imaging, Glycoproteomics, Two Tailed Test