Fig 1.
Timeline for 13C-tracer experiments with 3T3-L1 adipocytes.
Fig 2.
3T3-L1 adipocyte differentiation.
(A) Phase contrast images of 3T3-L1 cells from induction (day 0) to ten days post-induction. Cells initially display a fibroblast phenotype. Over the course of differentiation, cell morphology changes and cells accumulate lipid droplets internally. (B) Triglyceride staining of 3T3-L1 cells with Oil Red O. Initially, the fibroblast phenotype displays negligible staining. As the adipocytes mature, the amount of staining increases until almost the entire cell volume is stained red.
Fig 3.
(A) Glucose, lactate, and glutamine concentration profiles over the course of the tracer experiment between days 6 and 7 (mean ± stdev; n = 4 biological replicates). (B) Profile of lactate concentration versus glucose concentration.
The yield of lactate on glucose decreased during the experiment as indicated by a change in the slope.
Fig 4.
Amino acid concentration profiles over the course of the tracer experiment between days 6 and 7 (mean ± stdev; n = 4 biological replicates).
Valine, leucine and isoleucine were consumed at a high rate, while glycine and alanine were secreted by the cells.
Table 1.
Glucose, lactate and amino acid production and consumption rates (nmol/106 cells/h, mean ± SEM, n = 4 biological replicates).
Fig 5.
Total ion chromatogram from GC-MS analysis of fatty acid methyl esters (FAMEs).
Fatty acids were extracted from differentiated 3T3-L1 adipocytes. Selected ion recording of molecular ions was conducted for GC-MS analysis.
Fig 6.
Mass isotopomer distributions of (A) methyl pentadecanoate (C15:0) and (B) methyl palmitate (C16:0) for four parallel labeling experiments with [U-13C]valine, [U-13C]leucine, [U-13C]isoleucine and [U-13C]glutamine tracers.
Fig 7.
GC-MS analysis of fatty acid picolinyl ester derivatives.
(A) Chemical structure of fatty acid methyl ester derivatives. (B) Mass spectrum of palmitic acid (C16:0) methyl ester derivative. Intermediate mass fragments cannot be used for positional labeling information as they are not unique fragments. (C) Chemical structure of fatty acid picolinyl ester derivatives. (D) Mass spectrum of palmitic acid (C16:0) picolinyl ester derivative. The dominant fragments in the spectrum are separated by 14 amu corresponding to a unique loss of consecutive CH2 groups.
Fig 8.
(A) Fragments of pentadecanoatic acid (C15:0) measured by GC-MS. An asterisk (*) indicates the expected location of 13C-labeled carbon atoms from [U-13C]valine. (B) Mass isotopomer distributions for four fragments of C15:0 retaining different parts of the fatty acid carbon backbone (from [U-13C]valine experiment). (C) Mass isotopomer distributions measured for four fragments of C16:0 from [U-13C]valine experiment.
Fig 9.
Isotopomer spectral analysis (ISA) results.
(A) Estimated D(AcCoA)-values, fractional contributions to lipogenic acetyl-CoA precursor pool. (B) Estimated D(PropCoA)-values, fractional contributions to lipogenic propionyl-CoA precursor pool. (C) Estimated g(24h)-values, fractional new synthesis of each fatty acid during the 24 hr tracer experiment between days 6 and 7. (D & E) Estimated mass isotopomer labeling (M+1, M+2 and M+3) of AcCoA and PropCoA precursor pools from the various 13C-tracers used in this study.
Fig 10.
Pathways for branched chain amino acid catabolism.
Valine degradation produces one propionyl-CoA, isoleucine catabolism produces one propionyl-CoA and one acetyl-CoA, and leucine catabolism produces three acetyl-CoA. Colors indicate the carbons which arise in propionyl-CoA (blue), acetyl-CoA (red), and acetoacetate (green).