Fig. 2: Evaluation of overfeeding-induced changes in potential modulators of energy balance and their role in the response to overfeeding.

a–d Blood glucose (a, n = 5 per group), plasma insulin (b, n = 5 per group), plasma leptin (c, n = 5 per group), and plasma ghrelin (d, n = 4 Control d14, n = 6 Control d14 + 3, n = 2 ExpOF d14, n = 3 ExpOF d14 + 3) levels at different time points during the overfeeding intervention. e Fgf21 expression in eWAT, iWAT, iBAT, and liver (n = 6). f Plasma FGF21 levels (n = 6). g Schematic overview of the ExpOF setup in chow-fed FGF21 KO mice. Created with BioRender.com. h Body weight changes (percentage over baseline) in control (n = 4) and ExpOF (n = 5) FGF21 KO mice. Body weight is set at 100% at d0 (start of ExpOF). i Absolute body weight of same mice shown in h on day 0, 14, and 21. j Daily total (light pink for control mice, dark pink for ExpOF mice) and voluntary (purple for ExpOF mice) energy intake of same mice shown in h. k Heatmap showing average changes of top 20 regulated proteins (sorted by adjusted p value) between control and ExpOF mice at d14 and d14 + 3 (n = 6). Heatmap shows fold-changes in normalized protein expression relative to control d14 mice. Red indicates high relative protein abundance and blue indicates low relative protein abundance. Regulated proteins (FDR < 0.1) are highlighted in bold. l–o Effects on food intake (l, n), and body weight (m, o) in C57BL/6 J DIO male mice injected once with 1 mg/kg of DLL1 (l, m) or LGMN (n, o) human recombinant proteins or vehicle (n = 8 per group). p, q Open field test running traces (p) and total distance (q) of chow-fed lean C57BL6/J mice injected with LGMN (1 mg/kg) or vehicle (n = 10 per group). Data shown as mean ± SEM with individual values plotted (d–f, I, m, o, q). P values were calculated using 2-way ANOVA using overfeeding and time as factors (a–f, h–j, l, n), or unpaired two-tailed Welch’s t test (m, o, q). */**/*** was used when p < 0.05, p < 0.01, p < 0.001, respectively, in Welch’s t test or post hoc comparison after ANOVA (a–c, i, l–o). */#/$; **/##/$$; ***/###/$$$ were used when p < 0.05; p < 0.01; p < 0.001, for overfeeding/time/interaction effects, respectively (d–f). ns: non-significant. n: number of mice (biological replicates). Source data are provided as a Source Data file.