Fig. 4: Male-biased glycolytic activity and Pfkfb3 expression.

a The glycolytic pathway is illustrated, with enzymes shown in gray boxes and intermediate substances shown in open boxes. PFKFB3 mediates the reaction from F-6-P to F-2,6-BP, the latter of which acts as a strong activator of PFKM. b Male (n = 3) and female (n = 3) muscle fibers were prepared from type IIB-enriched regions of the quadriceps muscle (Supplementary Fig. 3a). The ECARs of cultured muscle fibers were examined, and the data were corrected by the ratio of the mean protein content in the fibers from the two sexes (male/female = 1.10) (Supplementary Fig. 3d). **p < 0.01. c The expressions of glycolytic genes were determined in the 10 experimental mouse groups (Supplementary Fig. 2) by qRT-PCR (n = 3 each group). Among paralogous genes, if any, the gene showing the highest expression was examined. d The expression of Pfkfb3 was extracted from the transcriptome datasets. e The expression of Pfkfb3 mRNA was determined by qRT-PCR (n = 3 each group). f The amounts of PFKFB3 protein were analyzed by western blotting. Whole proteins prepared from the type IIB-enriched areas of the quadriceps muscles of male (M) and female (F) were used. HeLa cell lysate was used as a control (C). Western blot images for PFKFB3 (upper left) and GAPDH (lower left) are shown. Full blot images are shown in Supplementary Fig. 5a. Three biologically independent samples were analyzed. The data were normalized to GAPDH and are presented as means ± SD (right). **p < 0.01. For c and e, the bars (means ± SD) with the same letter are not significantly different from each other (p < 0.01).