Extended Data Fig. 9. Fasting increases hypusination of eIF5A.

(a) Hypusine levels are increased after 24 hours -N in yeast BY4741 GFP-Atg8 cells, but not in ∆spe1. Representative immunoblot. (b) Quantification of [A]. N = 7(∆spe1 -N), 8(rest) biologically independent samples (yeast cultures). (c) Representative immunoblot of WT and ∆spe1, with and without 100 µM SPD after 6 hours -N, assessed for hypusine and GAPDH. (d) Quantification of [C]. N = 6 biologically independent samples (yeast cultures). (e) SPD supplementation does not further increase hypusination in BY4741 GFP-Atg8 yeast. Representative immunoblot. (f) Quantification of [E]. N = 4 biologically independent samples (yeast cultures). (g) Representative maximum projection images of confocal microscopy images of male w¹¹¹⁸ fly central brain regions probed for eIF5A and hypusine by immunofluorescence. Prior to dissection, the flies were fasted for 0 (ad lib) or 12 hours, starting at 8 pm. Scale bar = 50 µm. (h) Quantification of signal intensities as shown in [G]. N = 30 fly brains. (i) Representative immunoblot of female Drosophila w¹¹¹⁸ head extracts, assessed for hypusine and actin after 12 and 24 hours overnight fasting. RF = 12 hours refeeding. (j) Quantification of [I]. N = 6 biologically independent samples (fly head lysates). (k) Representative immunostaining microscopy images against hypusine after 12 hours fasting of female WT w¹¹¹⁸ and Odc1^MI10996/+ fly brains. Scale bar = 50 µm. (l) Quantification of [K]. N = 11(ad lib), 12(fasted) fly brains. (m) Immunoblots of cardiac tissue from young male transgenic IGF1R^tg or dnPI3K mice and their age-matched WT controls. Heart lysates were assessed for hypusine, total eIF5A and GAPDH. (n) Quantification of [M]. N = 6(WT), 7(IGF1R^tg), 3(dnPI3K) mice. (o) Representative immunoblot of human U2OS cells starved for 6 hours and assessed for hypusine, total eIF5A and GAPDH. (p) Quantification of [O]. N = 6 biologically independent samples. (q) Hypusine levels after five days of fasting in isolated human PBMCs of cohort 4, stratified by sex. N = 12(female baseline), 11(female fasted), 5 (male baseline), 4(male fasted) volunteers. (r-t) eIF5A^H levels after five days of fasting in isolated human PBMCs of cohort 4 do not correlate with age, pre-fasting baseline BMI or body weight loss (body weight ratio). N = 15 volunteers. Statistics: [B,D,L,NP,Q] Two-way ANOVA with Holm-Šídák’s multiple comparisons test. [F] One-way ANOVA with Holm-Šídák’s multiple comparisons test. [H,J] Two-tailed Student’s t-test with Holm-Šídák’s multiple comparisons test. [R-T] Simple linear regression. Bar graphs show the mean ± s.e.m. Source numerical data and unprocessed blots are available in source data.

Source data