Figure 1. Mammalian adipocytes synthesize mmBCFAs via fatty acid synthase.

a. mmBCFA abundance in differentiating 3T3L1 adipocytes. b. mmBCFA abundance in 3T3L1 adipocytes differentiated for 7 days +/− 500 nM B12. Two-tailed students t-test was performed on three cellular replicates (p=0.000019) with no adjustment for multiple comparisons. c. Atom-transition map demonstrating isotope incorporation into de novo synthesized fatty acids from [U-¹³C₆]glucose. Closed circles indicate ¹³C carbon. d. Isotopologue distribution of iso-C16:0 from [U-¹³C₆]glucose traced 3T3L1 cells. e. % enrichment from [U-¹³C₆]isoleucine-derived acetyl-CoA (M1-M2 only) in 3T3L1 adipocytes +/− 100 nM ND646 for 24 hours. f. Atom-transition map depicting transfer of isotope [3-²H]glucose through glycolysis, the pentose phosphate pathway and reductive biosynthesis. Open circles indicate carbon and small red circles indicate deuterium label from [3-²H]glucose. g. Contribution from [3-²H]glucose labelled NADPH to fatty acid de novo synthesis determined via ISA. 3T3L1 adipocytes cultured in tracer for 72 hours. h, Iso-C16:0 levels as % total FA in pooled CRISPR/Cas9 FASN KO 3T3L1 adipocytes following addition of isobutyrate for 24 hours. i. Relative abundances of mmBCFAs and C16:0 in differentiated 3T3L1s following addition of isobutyrate for 24 hours. Two-tailed students t-test was performed on three cellular replicates for each comparison with no adjustment for multiple comparisons. All data are presented as means ± SEM with dot plots overlaid except for g, where 95% confidence intervals from ISA model are shown. All data are representative of three cellular replicates, and each experiment was repeated 3 independent times with the exception of h, where two separate infections were carried out. The same result was obtained in each independent experiment. *p<0.05, **p<0.01, ***p<0.001.