Extended Data Fig. 3: Feimin is secreted in myofibers and myotubes.

a, Effect of serum on medium feimin levels in cultured myofibers of extensor digitorum longus (EDL) muscle from WT mice or feimin MKO mice. The myofibers were treated for 1 hr with 10% serum from feimin^−/− mice (feimin global knockout) after overnight fasting (fasted serum) or 1 hr refeeding (refed serum). n = 6 biological replicates. b, Effect of different glucose (Glc) doses on medium feimin levels in cultured myofibers of EDL muscle from WT mice or feimin MKO mice. The myofibers were treated in the presence or absence of glucose for 1 hr. n = 6 biological replicates. c, Effect of glucose (Glc, 10 mM) and/or insulin (INS, 100 nM) on medium feimin levels in cultured myofibers of EDL muscle from WT mice or feimin MKO mice. The myofibers were treated in the presence or absence of glucose and/or insulin for 1 hr. n = 6 biological replicates. d, Effect of serum on medium feimin levels in cultured myotubes from WT mice or feimin^−/− mice (feimin global knockout). The mature myotubes were treated for 1 hr with 10% serum from feimin^−/− mice after overnight fasting (fasted serum) or 1 hr refeeding (refed serum). n = 6 biological replicates. e, Effect of different glucose (Glc) doses on medium feimin levels in cultured myotubes from WT mice or feimin^−/− mice. The mature myotubes were treated in the presence or absence of glucose for 1 hr. n = 6 biological replicates. f, Effect of glucose (Glc, 10 mM) and/or insulin (INS, 100 nM) on medium feimin levels in cultured myotubes from WT mice or feimin^−/− mice. The mature myotubes were treated in the presence or absence of glucose and/or insulin for 1 hr. n = 6 biological replicates. g, Effect of glucose (Glc, 10 mM) and/or insulin (INS, 100 nM) on feimin mRNA in cultured myotubes from WT mice or feimin^−/− mice. The mature myotubes were treated in the presence or absence of glucose and/or insulin for 1 hr. n = 3 biological replicates. h, Effect of secretion inhibitors on medium feimin levels in cultured myotubes. Mature myotubes were pretreated with or without Brefeldin A (5 µg ml⁻¹), Glibenclamide (50 µM), GW4869 (10 µM), or Manumycin A (10 µM) for 30 min, followed by treatment with glucose (10 mM) and insulin (100 nM) for 1 hr. n = 6 biological replicates. i, Immunoblots showing that feimin is associated with exosomes. Purified exosomes from myotubes were digested with (PK + ) or without (PK-) Proteinase K. CD81 is a transmembrane marker protein of exosomes, and TSG101 is a lumenal marker protein of exosomes. TCL, total cell lysate. j, Effect of mTOR inhibitor (Torin 1, 250 nM), AMPK activator (A-769662, 10 µM), or AMPK inhibitor (Compound C, 10 µM) on medium feimin levels. Matured myotubes were pretreated with or without these chemicals for 30 min, followed by treatment with glucose (10 mM) and insulin (100 nM) for 1 hr. n = 6 biological replicates. k, Model showing the effect of glucose and insulin on feimin secretion. When glucose and insulin levels are high, mTOR activity is stimulated while the activity of AMPK, which has an inhibitory role in feimin secretion, is dampened. When glucose and insulin levels are low, mTOR is downregulated and AMPK is activated, thus reducing feimin secretion. Data are shown as mean ± s.e.m. Statistical comparisons were performed using two-way ANOVA followed by Tukey’s test (a-h, j). All individual points and P values are shown.

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