Gw1516

Lactic acid bacteria–derived fatty acids were produced as described previously (Fig. S1 and Table S1) (13 (link)). GW7647 and GW1516 were purchased from Sigma–Aldrich. The following antibodies were purchased from commercial sources: anti-HMGCS2 and anti-CPT1A (ab137043 and ab128568); anti-β-actin (A5441). All other chemicals of analytical grade were purchased from Sigma–Aldrich, FUJIFILM Wako Pure Chemical, or nakalai tesque.

Glucose and palmitate oxidation were determined in primary human skeletal muscle cells as described^{42 (link)}. Glucose oxidation was determined by incubation of cells with D-[U-¹⁴C]glucose in the absence or presence of 1 µM FCCP (Sigma-Aldrich). Plates were sealed and incubated for 4 h. Thereafter, media was acidified (1:8 vol 2 M HCl) and the liberated ¹⁴CO₂ was collected for 1 h in a center well containing 2 M NaOH. The amount of liberated ¹⁴CO₂ was determined by scintillation counting. Palmitate oxidation was determined following pre-treatment of cells with or without 10 nM GW1516 (GW501516, Sigma-Aldrich) for 96 h. Cells were incubated with 25 µM non-labeled palmitic acid and [9,10-³H]palmitic acid for 6 h. The amount of ³H₂O released into culture media was determined by scintillation counting. Results were normalized to total cellular protein content (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific) and scintillation counting was determined using a 1414 WinSpectral Liquid Scintillation Counter.

Agonists GW0742 and GW1516 were purchased from Sigma-Aldrich (Taufkirchen, Germany). Nano-HPLC solvents were LC-MS grade (VWR, Darmstadt, Germany), water was purified with a TKA X-CAD system (Thermo Fisher Scientific, Bremen, Germany). Trypsin (cleaving C-terminally of lysine and arginine), GluC (cleaving C-terminally of glutamate and aspartate), and ProTEV Plus (cleaving C-terminally of ENLYFQ(G/S)) were obtained from Promega (Mannheim, Germany). The cross-linker BS²G-D₀/D4 was obtained from Thermo Fisher Scientific (Rockford, IL). The urea-linker was synthesized and purified in-house.[13 (link)] Bpa was obtained from Bachem (Bubendorf, Switzerland). Tryptone, yeast extract, antibiotics, TCEP, and IPTG were purchased from Roth (Karlsruhe, Germany). Iodacetamide, DTT, D-desthiobiotin, DMTMM, and all other chemicals (Sigma-Aldrich, Taufkirchen, Germany) were obtained at the highest available purity.