Fig. 2.

The main active metabolite in adipocyte-conditioned medium is a small, hydrophilic metabolite that is different from 3-HIB. (A) Treatment of ICM with charcoal, boiling and filtering did not appreciably reduce the potency of ICM. (B) 3-HIB concentrations in adipocyte CM and ICM at the indicated times. n=4 biological replicates for the 1–12 h timepoints, and n=6 for 24 h and 30 h timepoints. DMEM+I, non-conditioned medium with equivalent concentration of added insulin. (C) Inhibition of BCAT increased levels of BCAAs and decreased levels of 3-HIB in ICM. (D) Inhibition of BCAT in adipocytes did not suppress the ability of ICM to induce FA uptake (ns, not significant). Data in A–D are means±s.e.m. n=3 biological replicates for each condition in A, C and D. Each experiment was replicated at least twice. **P<0.01, ***P<0.001, ****P<0.0001 (for ICM compared to DMEM control in A, CM compared to DMEM+I control in B, and BCATi compared to vehicle control in C); ^#P<0.05, ^##P<0.01, ^####P<0.0001 for ICM compared to DMEM+I control in B (one-way ANOVA with Dunnett's test for multiple comparisons). RFU, relative fluorescence units.